Electrochemical affinity biosensor system and methods

ABSTRACT

The present invention provides novel osmium-based electrochemical species for the detection of wide variety of analytes using immunological techniques. The present invention also provides diagnostic kits and test sensors supporting electrode structures that can be used with the osmium-based electrochemical species. The test sensor can be fabricated to support interdigitated arrays of electrodes that have been designed to provide amplification of the electrical signal amplification desired to analyze analytes that may be present at low concentrations.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. patent application Ser.No. 10/555,138 filed Sep. 29, 2006 which is a national stage of theInternational Patent Application PCT/US2004/21187 filed Jul. 1, 2004(which was published in English), which claims the benefit of U.S.Provisional Application No. 60/484,096 filed Jul. 1, 2003, all of whichare hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates to a system, reagents, and methods fordetecting analytes in fluids. More specifically, but not exclusively,the present invention is directed at electrochemical immunoassaysystems, and reagents for detecting and analyzing analytes in fluidsamples and methods thereof.

Electrochemical biosensors have been used in in vitro diagnostics fordetermining the presence and concentration of certain biologicallysignificant analytes in biological samples such as blood, urine, andsaliva. Diabetic blood glucose monitoring has been one of the mostcommon and successful commercial applications of electrochemicalbiosensors. Other diagnostics biosensor applications have been developedand include lactate, cholesterol, creatinine, blood gases, andelectrolytes. Both AC and DC electrochemical measurement techniques areemployed including amperometry, potentiometry, coulometry, andimpedance. The majority of the current biosensor technology relies onselected, free enzymes as a bio-recognition element for the analytes.Further, this technology typically can accurately measure a relativelyhigh concentration of the analytes in the mM range. Consequently,electrochemical detection can be accomplished using a macro electrodewithout the use of amplification techniques.

Other analytes of interest are found in much lower concentrationscompared to glucose. Such analytes include: drugs of abuse, such as,amphetamine, cocaine, phencyclidine (PCP), and tetrahydrocannabinol(THC); therapeutic agents, such as, theophylline, digoxin, digitoxin,and methotrexate; environmental pollutants, such as, PCB and atrazine;biowarfare agents, such as, anthrax, botulism, and sarin; proteins; andhormones.

Various affinity-base assay techniques that use labels have beenexplored to detect these analytes. The affinity-based techniques includethe use of: enzyme labels, radioisotopic labels, chemilluminescentlabels, fluorescent labels, and electrochemical redox labels. However,many of these techniques are labor intensive requiring many steps thatare best performed in a laboratory by skilled technicians. The numberand complexity of steps prohibit routine use of these techniques “in thefield”. Many of these tests utilize variations on the competitive EnzymeLinked Immunosorbent Assays (ELISA). Examples include atrazine assaysfrom Strategic Diagnostics and EnviroLogix, Inc. both of which have manymanual steps including a 15 minute and 1 hour incubation timerespectively. Similar ELISA-based assays and other immunoassay formatswill be found that can be applied to a diverse set of assay across manyindustries but few are capable of a rapid onsite quantitative assay. Oneof the most commonly available immunoassay formats used for rapidtesting or point of care devices is known as lateral flow assays andutilizes immunochromatography. Most of these products are “screeningassays” that provide a qualitative result (positive/negative) indicatedby the presence or absence of a line. Results are often visually readand often hard to interpret when minor or partial lines are present.Most of these assays require follow-up with another method such as GC/MSor HPLC if the result is positive. There is a great need to provide atechnology to these diverse industries to allow rapid affinity-baseddetection. Fast detection allows rapid actionable results.

The use of electrochemical redox labels, which are also referred to aselectron transfer agents or electrochemical mediator labels, have beenshown to provide practical and dependable results in affinity-basedelectrochemical assays. However, the use of electrochemical detectiontechniques for quantifying the redox labels and, consequently,correlating the concentration of the redox labels with the analyteconcentration, has not been without problems. Electrochemicalmeasurements are subject to many influences that affect the accuracy andsensitivity of the measurements, including those related to the properselection of the mediator conjugate to variations in the electrodestructure itself and/or matrix effects derived from variability of thesamples.

U.S. Pat. No. 5,589,326 and WO 96/25514 disclose mononuclear osmiumcomplexes comprising two bidentate ligands and one imidazole bound viaits ring-nitrogen atoms to the central osmium. These osmium complexescan be used as redox mediators especially in electrochemical biosensors.Nakabayashi et al., Sensors and Actuators B 66 (2000):128-130 examinedthe evaluation of Os (II) complexes as mediators accessible forbiosensors. Mononuclear Os (II) complexes were synthesized and the redoxpotentials of Os (III/II) complexes could be lowered by the use of4,4′-dimethyl-2,2′-bipyridine, imidazole and chloride ion as ligands. US2003/0096997 describes mononuclear transition metal complexes and thereuse as redox mediators. As metal atom cobalt, iron, ruthenium, osmium,or vanadium is used. Two bidentate ligands and two other ligands arebound to the central metal. Csöregl et al., Anal. Chem. 1994, 66,3131-3138 discloses a glucose-sensing layer made by cross-linkingglucose oxidase with a polymer derived of poly (vinylimidazole), made bycomplexing part of the imidazoles to [Os (bipyridine) 2Cl]^(+/2+).

Many immunoassays require a detection limit much lower than what iscurrently possible with the electrochemical detection on a conventionalmacro-electrode. Therefore, signal amplification techniques must be usedfor these assays to significantly improve the electrochemical detectionlimit.

In light of the above-described problems, there is a continuing need foradvancements in the relevant field, including improved systems, methods,compositions, and reagents related to enhancing the detection analysisof various analytes including therapeutic drugs, drugs of abuse, diseasestate, analytes for food testing, analytes of environmental importance,and biowarfare agents. The present invention is such an advancement andprovides a variety of benefits and advantages.

SUMMARY OF THE INVENTION

In one form, the present invention provides novel osmium-basedelectrochemical species that can be used in immunoassays. The osmiumspecies can be coupled to a specific binding ligand to detect analytesof interest. The osmium species can include 1, 2, or 4 osmium centersthat are coupled to the specific binding ligands using a variety oflinking groups. The linking groups can be selected for specific types ofanalytes or to accommodate the different properties exhibited by theanalytes. For example, the linking group can be selected to impartdifferent degrees of hydrophilicity (or conversely hydrophobic)properties.

The novel osmium-based electrochemical species can be used to detect andanalyze a variety of analytes of interest, for example, biowarfareagents, therapeutic agents, environmental pollutants, proteins, andhormones.

The osmium-based electrochemical species can be used in conjunction withdifferent test sensors and diagnostic kits. In one form, theosmium-based electrochemical species are used in a homogenousimmunoassay to detect the analytes of interest. The assay techniquesaccording to the present invention can be used with different testsensors and meters. In particularly preferred embodiments, the assaytechniques can be used to analyze samples that contain a particularlylow concentration of the desired analyte. In other embodiments, theassay techniques can be used to provide reliable assay results within avery short test time—preferably less than about 10 seconds.

In one form, the present invention provides novel test sensors thatinclude interdigitated arrays of electrodes. The electrode arrays caninclude first and second working electrodes, as well as counter andreference electrodes. A bipotentiostat can be used to control differentvoltage potentials between the various combinations of working andreference (or counter) electrodes.

In another form, the present diagnostic kits can include portable testdevices that can be readily used “in the field”. The portable testdevices can include the test sensors, a configurable, portable meter,and, optionally, a sample collection chamber.

Further objects, features, aspects, forms, advantages, and benefitsshall become apparent from the description and drawings containedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatical representation of one embodiment of adiagnostic kit in accordance with the present invention.

FIG. 2 is a perspective view of one embodiment of a test sensor having aplanar array of electrodes useful for detecting and analyzing for aplurality of analytes of interest in accordance with the presentinvention.

FIG. 3 is a perspective view of another embodiment of a test sensorhaving a planar array of electrodes useful for detecting and analyzing aplurality of analytes of interest with a plurality of sample ports inaccordance with the present invention.

FIG. 4 is an exploded view of yet another embodiment of a test sensoruseful for detecting and analyzing a plurality of analytes.

FIG. 5 is a perspective view of the test sensor of FIG. 4.

FIG. 6 is a test sensor configured for multi-analyte testing that can bedipped into the sample.

FIG. 7 is a perspective view of a test sensor and meter configured forsingle assay testing that can be dosed with blood from a finger prickedwith a lancet.

FIG. 8 shows a perspective view of a test sensor and meter configuredfor a single assay that can be dosed with a pipette.

FIG. 9 shows a perspective view of a meter and test sensor configuredfor multi-analyte testing which includes the assay attached to a samplecollection chamber.

FIG. 10 is a partial cross-sectional view a pair of electrodesillustrating the conditions of steady-state current limited by diffusionof a reversible mediator (M) which is alternatively oxidized and reducedon the interdigitated electrode fingers.

FIG. 11 is an enlarged plan view of a planar interdigitated array (IDA)electrode set suitable for measuring redox cycling of reversiblemediators in accordance with the present invention

FIG. 12 is a partial cross-sectional of a vertical interdigitated arrayelectrode set for reversible mediator measurement in accordance with thepresent invention.

FIG. 13 is an enlarged plan view of a vertical interdigitated arrayelectrode of FIG. 12.

FIG. 14 is a three-dimensional plot of an electrical current vs.electrode dimensions for a planar IDA normalized for physical electrodearea.

FIG. 15 provides Table 3 listing various embodiments of IDAs and VIDAsprepared and evaluated in accordance with the present invention.

FIG. 16 is a graph illustrating the ability to increase the currentamplification by decreasing the gap width between the electrodes in anIDA in accordance with the present invention.

FIG. 17 is a diagram illustrating one embodiment of a sequential bindingassay in accordance with the present invention.

FIG. 18 illustrates one synthetic scheme for the preparation of anOs(bipyridyl)histamine electrochemical label in accordance with thepresent invention.

FIG. 19 illustrates a synthetic scheme for the preparation of anosmium-amphetamine conjugate in accordance with the present invention.

FIG. 20 illustrates a synthetic scheme for the preparation of anosmium-theophylline conjugate in accordance with the present invention.

FIG. 21 illustrates a synthetic scheme for the preparation of anosmium-PCP conjugate in accordance with the present invention

FIG. 22 illustrates a synthetic scheme for the preparation of anosmium-THC-2 conjugate in accordance with the present invention.

FIG. 23 illustrates a synthetic scheme for the preparation of anosmium-THC-1 conjugate in accordance with the present invention.

FIG. 24 illustrates a synthetic scheme for the preparation of anosmium-methotrexate conjugate in accordance with the present invention.

FIG. 25 illustrates a synthetic scheme for the preparation of anaromatic trifluoroacetamido protected linker for use in accordance withthe present invention.

FIG. 26 illustrates a synthetic scheme for the preparation of adi-osmium aromatic trifluoroacetamido and mono osmium aromatictrifluoroacetamido protected linker or electrochemical label inaccordance with the present invention.

FIG. 27 illustrates a synthetic scheme for the preparation of adi-osmium electrochemical label with an aromatic linker in accordancewith the present invention.

FIG. 28 illustrates a synthetic scheme for the preparation of adi-osmium THC-1 conjugate in accordance with the present invention.

FIG. 29 illustrates a synthetic scheme for the preparation of adi-osmium electrochemical label with an aliphatic linker in accordancewith the present invention.

FIGS. 30 and 31 illustrate a synthetic scheme for the preparation of anosmium-PEG (linker) electrochemical label in accordance with the presentinvention.

FIG. 32 illustrates a synthetic scheme for the preparation of an osmiumPEG THC-2 conjugate in accordance with the present invention.

FIG. 33 illustrates a synthetic scheme for the preparation of an osmiumPEG methotrexate conjugate in accordance with the present invention.

FIG. 34 illustrates a synthetic scheme for the preparation of a tetracarboxylic acid linker group in accordance with the present invention.

FIG. 35 illustrates a synthetic scheme for the preparation of theprotected precursor of the tetra-osmium trifluoroacetamidoelectrochemical label in accordance with the present invention.

FIG. 36 illustrates a synthetic scheme for the deprotection of the tetracarboxylic acid linker of a tetra-osmium electrochemical label inaccordance with the present invention.

FIG. 37 illustrates a synthetic scheme for the preparation of an osmium(dimethyl biimidazole)₂ histamine linker or electrochemical label inaccordance with the present invention.

FIG. 38 is a CV spectrum of an osmium-theophylline conjugateelectrochemical label.

FIG. 39 is a plot illustrating the steady state response of theosmium-theophylline conjugate electrochemical label.

FIG. 40 is a plot of the dose response of the osmium-theophyllineconjugate electrochemical label.

FIG. 41 is a plot of the antibody inhibition of the osmium-theophyllineconjugate electrochemical label.

FIG. 42 is a plot of a theophylline assay response in a serum matrix.

FIG. 43 is a CV spectrum of an osmium-amphetamine conjugateelectrochemical label.

FIG. 44 is a recycling CV of the osmium-amphetamine conjugateelectrochemical label.

FIG. 45 is a plot of the conjugate response of osmium-amphetamineelectrochemical label.

FIG. 46 is an assay curve for amphetamine in PBST obtained in thepresence of the osmium-amphetamine electrochemical label.

FIG. 47 is a recycling CV for bis(2,2′-bipyridyl)imidazole chloro osmium(III)dichloride label on a 2 μM gap/width interdigitated array electrodecontaining 750 interdigitated electrode pairs.

FIG. 48 is an osmium biotin conjugate dose response on a 2 μm IDAelectrode.

FIG. 49 is a plot of the steady-state response recorded at 0.5, 2, and10 seconds after sample introduction for a biotin assay on a 2 μm IDAelectrode.

FIG. 50 is a plot of current vs. time of the steady-state response ofrepresentative concentrations of the biotin assay of FIG. 49.

FIG. 51 is a CV spectrum of the mono-osmium aromatic trifluoroacetamidoprotected linker.

FIG. 52 is a CV spectrum of the di-osmium aromatic linkerelectrochemical label.

FIG. 53 is a graph comparing the dose response curve of a di-osmiumlinker, a mono-osmium linker, and bis(2,2′-bipyridyl)imidazole chloroosmium (III) dichloride.

FIG. 54 is a CV spectrum of the di-osmium-THC-1 conjugate.

FIG. 55 is a graph of the response of osmium-PEG-THC-2 conjugate.

FIG. 56 an enzyme amplified plot of the conjugate response ofosmium-PEG-THC-2 electrochemical label with and withouthydroxypropylbetacylcodextrin.

FIG. 57 is a CV spectrum of the osmium-PEG-methotrexate conjugate.

FIG. 58 is a graph of the dose response of osmium-PEG-methotrexateconjugate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a variety of techniques and systems foranalysis of various analytes. The techniques can employ novelelectrochemical mediators in conjunction with selected specific bindingpartners for the analytes of interest. The system can include a varietyof test sensors carrying different electrode configurations andchemistries to detect or analyze the desired analytes. Additionally, avariety of test meters and configurable platforms can be employed withthe test sensors to provide an accurate, reliable, and convenient-to-useassay technique.

When used herein, the following definitions define the stated term:

The term “electrode structure” refers to a combination of all activeelectrode areas that may have contact with the sample, the redoxreversible conjugates, and/or the osmium conjugate; the electrode tracesleading to the contact pads; and the contacts pads that allow anelectrical contact with a meter or other instrument.

The term “active electrode area” when used in conjunction with an IDAelectrode includes the electrode regions in contact with the sampleincluding a reference electrode and at least a first and second workingelectrode dimensioned to allow diffusional recycling of the diffusibleredox reversible conjugates in the sample when a predeterminedredox-reversible species dependent cathodic and anodic potential isapplied to the working electrodes.

In the case of enzyme amplification the “active electrode area” includesthe electrode regions in contact with the sample including a referenceelectrode and at least a first working electrode.

The term “IDA electrode” refers to an Interdigitated Array electrodeoften drawn as a pair of comb-type electrodes but can include othershapes that bring two or more electrodes in close proximity to allow forredox recycling between the electrodes. Included in this definition areelectrodes that may be spatially separated in different planes alsoreferred to as Vertical Interdigitated Array electrodes (VIDA).

The term “working electrode” as used herein refers to an electrode wheremeasured events (i.e., oxidation and/or reduction) take place and theresultant current flow can be measured as an indicator of analyteconcentration.

The term “anodic potential” refers to the more positive potentialapplied to the anode, and “cathodic potential” refers to the lesspositive or negative potential applied to the cathode (vs. a referenceelectrode such as Ag/AgCl)

A “test sensor” refers to a combination of structures and reagentsincluding all subcomponents such as plastics, spacers, and adhesives aswell as the specific architectural components, such as, capillaries,measurement zones, and electrode structures. A test sensor can includethe required components structures, and reagents for a single assay orit may contain the components, structures and reagents needed formultiple assays. The test sensor of this invention may also include asample collection chamber and/or mixing chambers in addition to themeasurement zone.

The term “measurement zone” is the region of the test sensor in theredox reversible conjugates which is in contact with the activeelectrode area and capable of being interrogated during the assay.

This region of the configurable test sensor design should remainvirtually identical from assay to assay with the exception of the assayspecific reagents and IDA electrode dimensions. Multiple assay designswould have multiple measurement zones except in the case of usingmultiple redox mediators with varied redox potentials as described inU.S. Pat. No. 6,294,062.

“Interfering substances” include any species including the analyte ofinterest that elevates or reduces the signal desired from the analyte.Interferents can be inherently part of the sample matrix such asascorbic acid and uric acid that can be oxidized in blood or urine.Proteins or hydrophobic molecules such as THC can interfere withelectron transfer to the electrodes by forming a passivation layer onthe electrode surface reducing the expected response.

A “bipotentiostat” is the measurement engine that allows separate andindependent control of the potential of two working electrodes “WE1” and“WE2” in the same electrochemical cell along with the reference andcounter electrodes.

An “electrochemical label” as used herein refers to a chemical speciescapable of reversible oxidation and reduction in a liquid sample.Electrochemical labels can include complexes of transition metal ions,for example iron (ferrocene and ferrocene derivatives), ruthenium, andosmium. In preferred embodiments, the electrochemical label for thepresent invention is selected as an osmium organometallic species.

The “sample collection chamber” is the area first in contact with thespecimen containing the analyte. Examples include a capillary fill zone,cuvette, cup, or other sample receiving vessel to receive the samplecontaining analyte. The sample collection chamber as used herein is aregion that collects a volume of sample sufficient to subsequently runthe desired assays. The sample collection chamber may immediately passall or a portion of the sample to the sample receiving zone ormeasurement zone and run the assay, or it may hold the sample until thedevice is triggered at a later time to pass the sample to theappropriate zones. In selected embodiments, the sample collection,sample receiving, reaction chamber or zone, and the measurement zone areone and the same zone or region.

The “reaction chamber or zone” is the area in which the sample caninteract with the reagents. This can be a simple hydrating ordissolution of a single reagent or a sequential scheme of reacting withmultiple reagents. The sample receiving zone can facilitate mixing andcan pass the sample to the measurement zone. In at least one embodiment,the sample receiving zone is one and the same as the measurement zone.

The term “antibody” refers to (a) any of the various classes orsubclasses of immunoglobulin, e.g., IgG, IgM, derived from any of theanimals conventionally used, e.g., sheep, rabbits, goats or mice; (b)monoclonal antibodies; (c) intact molecules or “fragments” ofantibodies, monoclonal or polyclonal, the fragments being those whichcontain the binding region of the antibody, i.e., fragments; devoid ofthe Fc portion (e.g., Fab, Fab′, F(ab′)₂) or the so-called“half-molecule” fragments obtained by reductive cleavage of thedisulfide bonds connecting the heavy chain components in the intactantibody. The preparations of such antibodies are well-known in the art.

In general, the present invention is directed to the detection andanalysis of a wide variety of analytes. The analytes of interest can befound in a variety of sources, including humans, animals, plants, food,waste effluent, and ground water. The analytes may be of interestbecause they may be a therapeutic drug or abused substance whose in vivoconcentration and activity are of interest for the well being andtreatment of a patient. Other analytes of interest include analytes ofenvironmental interest which includes monitoring water and food suppliesfor pesticides, herbicides, or other contaminants.

The diagnostic technique of this invention uses an electrochemicalimmunoassay to detect and analyze the analytes. The preferredimmunoassay uses an electrochemically detectable label. In a preferredembodiment, the label is detected by measuring the current generated asthe label undergoes multiple oxidation reduction cycles at or on theelectrodes. Typically, the current generated by the oxidation/reductionof the detectable label is quite small and must be amplified to allowfor an accurate and repeatable analysis of the desired analyte. Thecurrent can be amplified by diffusional recycling under steady stateconditions and/or enzyme recycling.

The detection and analysis of the analytes can be conducted using a testkit that includes various components. Minimum components include ameter, test sensor, and sample. Preferably, a portable handheld meterconfigured to work with specific test sensor assays can simplify theassay method. In one embodiment, the meter is a portable handheldbipotentiostat designed for easy configurational changes to various testsensors. In another embodiment, the meter consists of a commerciallyavailable PDA or other portable computer device and a bipotentiostatmodule that plugs into or attaches to the device. With thisconfiguration, software changes can be used with a variety of testsensor assays to configure the use of the same module to create assaysfor a diverse array of products and markets. The analysis is conductedwith a small sample volume from about 4 μL to about 50 μL per assay.Sample collection volumes for the test sensor will vary depending onwhat is practical for the application. Blood collected from a lancetpricked finger will often be of volumes less than 15 μL but a urinesample collection device must conveniently handle larger volumes.Consequently, the test sensor configuration will vary but the underlyingelectrode structures and the measurement zones will in general remainthe same from test sensor to test sensor except for the assay specificreagents such as the electrochemical conjugate and the affinity bindingpartner (antibody).

The active electrode area of the electrode structure includes at least afirst working electrode, a second working electrode, a referenceelectrode, and a counter electrode. The first and second workingelectrodes are dimensioned to allow diffusional recycling with a redoxreversible conjugates in the sample when predetermined anodic andcathodic potentials are simultaneously applied to the workingelectrodes. Electrodes dimensioned to allow diffusional recycling aretypically in the form of arrays such as microdiscs, microholes, ormicrobands. In one embodiment, the electrodes are in the form of aninterdigitated arrangement of microband electrodes with micron orsubmicron spacing. When the distance between two differently polarizedelectrodes are sufficiently close, the diffusion layers aresuperimposed. Redox species oxidized at one electrode diffuse to and arereduced by the neighboring electrode. This results in an amplifiedcurrent signal due to the species being repeatedly oxidized and reduced.

The test sensor can contain a sample collection chamber and a samplereceiving chamber for receiving the liquid sample. The sample collectionchamber can include, for example, a capillary fill chamber, cuvette,cup, or other sample receiving vessel to receive the sample containinganalyte. In one embodiment, the sample collection chamber and samplereceiving chamber may be the same chamber. In yet another embodiment,the sample collection chamber, the sample receiving chamber, and themeasurement zone may be the same chamber. Embodiments with separatesample collection chambers or zones can be designed to collectefficiently and conveniently the preferred method of sample collectionfor a particular test sensor assay. Some preferred sample collectionmethods include a capillary chamber to pull in the blood from a fingerstick or a port introduction of the sample by other means includingdipping into a bulk sample or via a syringe or pipette. In anotherembodiment, the test sensor would include a larger sample collectionchamber such as a cup useful for the collection of groundwater, wasteeffluent, or urine. Test sensors with larger sample collection chambersare desired in various industries when it may be important to maintainadditional samples and/or seal the sample with a tamper resistant seal.This provides particular advantages for samples that may have legal,evidentiary issues or for samples suspected of containing biohazardcontaminants. Alternatively, the test sensor could contain only themeasurement zone serving also as the sample collection and samplereceiving zone. In all cases, the electrochemical immunoassay portion ofthe test sensor requires only a small sample sufficient to contact anddissolve with a predetermined amount of redox reversible conjugates anda specific binding partner.

The electrode structures can be supported on one or more walls of thechamber, wherein at least a portion of the electrode structure, theactive electrode area, is in contacted with the liquid sample. Thecontact regions of the electrode structure enable the meter ormeasurement module to apply the respective cathodic and anodicpotentials to the working electrodes to carry out the present method.The anodic and cathodic potentials are applied relative to the referenceelectrode—usually a Ag/AgCl ink using a bipotentiostat. The electrodestructure optionally will include a counter electrode for currentcontrol. The bipotentiostat is utilized to apply a first cathodicpotential to a first working electrode and a first anodic potential to asecond working electrode; the first cathodic and anodic potentialscorrespond to those respective potentials necessary to establish currentflow through the sample due to diffusional recycling of the first redoxreversible conjugates. Optionally, the potential on one workingelectrode can be set at a first diffusible species dependent anodicpotential and current flow is measured as the potential of the otherworking electrode is swept through a potential corresponding to thepredetermined diffusible species dependent cathodic potential (or viceversa).

The cathodic and anodic potentials appropriate for each reversible redoxspecies can be readily determined by empirical measurement such ascyclic voltammetry (CV). This technique was used to determine the redoxpotentials and reversibility of the electrochemical mediator and labels.In addition, a recycling CV was also used to measure the ability of anIDA electrode to recycle a known concentration of redox reversibleconjugates and determine the effective amplification. A recycling CV isperformed by fixing the first working electrode potential at either anoxidation or reduction potential and then scanning the second workingelectrode between oxidation and reduction. CVs and recycling CVs wereboth performed using a CHI 832A electrochemical detector from CHInstruments, Austin, Tex.

Preferred electrochemical mediators are redox reversible conjugatesselected for several attributes including one or more of the followinglow redox potentials, fast mediation kinetics, fast electron transferrate at the electrode surface, ease of analyte conjugation, stability,solubility, toxicity, and inhibition of the redox recycling upon pairingwith the specific binding partner (antibody). Bipyridyl osmium complexconjugates as discussed in U.S. Pat. No. 6,352,824 and imidazole-osmiumcomplex conjugates as discussed in U.S. Pat. No. 6,262,264 are bothexamples of mediators with appropriate properties. The mediators in theaforementioned patents generally meet the desired properties and can beviewed as a starting point in the selection of a mediator for assaydevelopment. Conjugates of mediators were prepared and evaluatedaccordingly as assays for various analytes of interest includingamphetamine, theophylline, cocaine, PCP, morphine, THC, andmethotrexate.

Although the previous class of electrochemical conjugates or labelsperformed well with most of the desired assays, certain electrochemicalconjugates do not. As an example, the following osmium histamine linkeddrug conjugates, osmium THC-2 (compound 17), osmium THC-1 (compound 19)and osmium methotrexate (compound 21), all met many of the desirableelectrochemical features but all suffered in terms of solubility andantibody recognition. The osmium methotrexate conjugate was not solublein the aqueous PBST matrix and required the addition of DMF. A ratio of30:70 DMF:PBST was used to solubilize the osmium methotrexate conjugate.To overcome these specific assay difficulties, additional conjugatestructures were proposed and synthesized. A longer, more flexible andhydrophilic linker, herein called PEG-linker, was purchased asO—(N-Boc-2-aminoethyl)-O—(N-diglycolyl)-2-aminoethyl hexaethylene glycol(compound 33) from Nova Biochem. The synthesis of an osmium-PEG-aminederivative is shown as compound 36 and derived using the synthesisschemes of FIGS. 30 and 31. This compound was then used to prepareelectrochemical labels for THC (compound 37) and methotrexate (compound38) as shown in synthesis schemes of FIG. 32 and FIG. 33 respectively.Difficulties with the THC and methotrexate assays were somewhat expecteddue to their hydrophobic nature, lower required detection limits, andavailable antibodies compared to other assays developed.

In addition to the hydrophilic PEG linker, a second useful conjugatetype was prepared to moderately improve the detection sensitivity.Osmium complexes with multiple redox centers were proposed. Syntheticschemes were prepared for 2 and 4 osmium redox centers per analytebinding site. It was expected that “D” the diffusion coefficient woulddecrease with these new conjugates due to the increase in the conjugatemolecular weight. But by doubling or quadrupling the available redoxsites, there was a potential for increased recycling.

The osmium-PEG-linkers for THC-1 and methotrexate achieved improvedsolubility in comparison with normal osmium the hydrophobic antigens.Both were able to be dissolved in a PBST matrix without the use of anorganic solvent as previously used. It is also suggested that thislinker may also achieve better accessibility to the antibody due to thelong flexible and hydrophilic nature of the linker. Theosmium-PEG-conjugates and the di-osmium conjugates performed reasonablywell in electrochemical characterization including CVs and conjugatedose response curves. These new mediator conjugates perform well in manyassays and offer specific improvements to overcome certain assaydifficulties associated with mediators being used in the art, such asmediator conjugates of hydrophobic antigens including, for example,tetrahydrocannibinol and methotrexate antigens.

In aqueous solutions, the useable range of potentials of the first andsecond working electrode can be selected to be about 600 mV to −600 mVvs. Ag/AgCl reference electrode to avoid oxidation or reduction ofwater. Electrochemical labels with lower redox potentials are preferredto avoid interference from possible oxidizable interferents such as uricacid and ascorbic acid. U.S. Pat. No. 6,294,062 discusses that multiplemediators of differing redox potentials mixed together can be measuredindependently of each other on an IDA electrode if the reversible redoxspecies are selected to be to have redox potentials differing by atleast 50 mV. Additionally, multiple analytes can be measured withmediators of similar or different potentials if the different reversibleredox species are separated or segregated into different measurementchamber. Measurement of steady state currents associated with a redoxrecycling of unbound mediator on an IDA electrode is proportionate tothe concentration of analyte. The current can be measured at WE1 WE2, orboth.

The present invention can be used to simultaneously measure two or moreanalytes in a single sample. In one preferred mode of operation, the kitincludes a series of electrode sets or structures; each set ofelectrodes are disposed within a separate sample chamber. The liquidsample is supplied to the separate sample chambers. For example, a testsensor can include at least a second sample chamber supporting a secondelectrode set configured as described above for the first electrode set.Additionally, the separate sample chambers can contain different redoxreversible conjugates.

The method includes detecting an analyte in a sample by measuring theconcentration of an unbound electrochemical label which is correlated tothe concentration of the desired analyte in the sample.

Table 1 shows possible detection ranges for analytes in comparison toblood glucose monitoring. Consequently, the diagnostic techniques shouldbe highly sensitive. Affinity-based assay techniques can provide thesensitivity to detect these analytes.

TABLE 1 Analyte Concentrations Ranges Typical Suggested AnalyteConcentrations Actionable values Test Range Glucose 2-6 mM >6 mM, <2.21-33 mM mM Theophylline 56-111 μM >138 μM 10-222 μM Amphetamine 220-230nM 3.5-7.0 μM 1-10 μM (Cutoff 6.7 μM) Morphine 180-700 nM 1.75-10.5 μM1-20 μM (Cutoff 1 or 7 μM) Cocaine 330-660 nM >3.3 μM 0.5-10 μM (Cutoff1.0 μM) Methotrexate Varies from (Post Dose) Varies over(Chemotherapeutic agent) time and >10 μM (24 hr) large range. amount ofdose >1 μM (48 hr) Multi assay to >0.1 μM (72 hr) cover specific rangesmay be best approach Tetrahydrocannabinol Not Applicable 160-640 nM50-1000 nM (Cutoff 160 nM) Oxycodone 48-127 nM >320 nM 50-1000 nMDigitoxin 13-39 nM >39 nM 2.6-85 nM (cardiac glycoside) Digoxin 1-2.6nM >3 nM 0.22-6.44 nM (cardiac glycoside) Atrazine (herbicide) <15nM >15 nM 0.5-25 nM Note: Cutoff concentration: The specificconcentration of drug or drug metabolite in the sample that is chosen asa limit to distinguish a positive from a negative test result. Cutofflevels are mandated for U.S. Federal Government employees but may varyfor workplace testing and in specific countries

Recently, there have been significant advances in electrochemicalaffinity biosensor technology that relies on the information obtainedfrom a complex between the analyte and a “specific binding partner”.Such techniques typically employ a labeled ligand analog of a targetanalyte, where a ligand analog is selected so that it bindscompetitively with the analyte to the specific binding partner. Theextent of binding of the labeled ligand analog to the specific bindingpartner can be measured and correlated with the presence and/orconcentration of the analyte in the sample. Examples of analytes andtheir specific binding partners are listed in Table 2 below.

TABLE 2 Analyte Specific Binding Partner Antigen (e.g., a drug) AntibodyAntibody Antigen Hormone Hormone Receptor Hormone Receptor HormonePolynucleotide Complementary Polynucleotide Strand Avidin Biotin BiotinAvidin Protein A Immunoglobulin Immunoglobulin Protein A LectinsSpecific Carbohydrates Carbohydrate Lectins

In one form of the present invention, the binding group of theelectrochemical label comprises an antigenic determinate, an epitope, ora ligand analog, typically, via one or more linker groups to form a“redox reversible conjugates” described above. The term “ligand analog”as used in the present invention includes within its meaning a chemicalspecies capable of complexing with the same specific binding partner asthe analyte being measured and can include the analyte itself. Lowmolecular weight species are most desirable in view of thediffusion-based electrochemical detection technique utilized in carryingout the present method. Consequently, it is desirable that the redoxreversible conjugate having a molecular weight of less than about 50,000Daltons, more preferably less than about 10,000 Daltons. Mostpreferably, the molecular weight of the redox reversible conjugate isbetween about 500 and about 5,000 Daltons.

Examples of ligand analogs for use in the present invention include, butare not restricted to: peptide hormones (e.g., thyroid stimulatinghormone (TSH), luteinizing hormone (LH), follicle stimulating hormone(FSH), insulin and prolactin) or non-peptide hormones (e.g., steroidhormones such as cortisol, estradiol, progesterone, and testosterone) orthyroid hormones such as thyroxine (T4) and triiodothyronine), proteins(e.g., human chorionic gonadotropin (hCG), carcino-embryonic antigen(CEA) and alphafetoprotein (AFP), drugs (both drugs for therapeutic use,drugs of abuse and/or regulated drugs), such as amphetamine, sugars,toxins or vitamins and biowarfare agents. Specific examples of ligandanalogs which can be included as ligand analogs in accordance with thepresent invention include, but are not restricted to: cocaine,amphetamine, morphine, barbiturate, theophylline, phenylcyclidine (PCP),tetrahydrocannabinol (THC), methotrexate, benzodiazepine, phenyloin,carbamazepine, phenobarbital, gentamicin, amikacin, vancomycin,tobramycin, procainamide, lidocaine, quinidine, valproic acid, digoxin,digitoxin, tricyclic antidepressants (TCA), such as: buprenorphine,amitrptyline, desipramine, imipramine, nortriptyline, doxepin,immunosuppressants. Warfare or biowarfare agents that can be includedwith the present invention include, but are not restricted to: racin,anthrax (B. anthracis.), small pox, botox, and botulinum toxin.

FIG. 1 is a diagrammatical illustration of a system or a diagnostic kit10 for detecting and/or analyzing one or more analytes in a samplefluid. Kit 10 includes a test sensor 12, a measurement module 14, and ahandheld or portable controller 16.

In the illustrated embodiment, test sensor 12 includes an electrodestructure 22. The electrodes in the electrode structure can be parallelto each other and supported on the same wall of the detection chamber oropposing each other with one electrode supported on one wall and anotherelectrode supported on an adjacent wall or an opposite wall of thedetection chamber. In a preferred embodiment, the electrode set includesa multi-array electrode such as an interdigitated array (IDA). Eachelectrode in the IDA includes a plurality of “fingers” whichinterdigitate with the “fingers” of the other electrode. The individualelectrodes in the IDAs can be either parallel to each other or opposingeach other. In other embodiments, the multi-array electrode can befabricated as a vertical interdigitated array electrode described morefully below.

In preferred embodiments, test sensor 12 provides a sequential analysisof one or more analytes in a sample solution. Preferably, the reagentssupplied onto test sensor 12 are provided in dry form with the fluid inthe test sample providing the medium for conducting the analysis.

Test sensor 12 can be provided as either a flexible strip or a rigidstrip discussed more fully below. The rigid test sensor can befabricated, for example, using integrated circuit technology on asilicon wafer.

Test sensor 12 includes a first end 26 and an opposite, second end 28. Asample port 30 is positioned on test sensor 12 adjacent first end 26. Inalternative embodiments, a sample port 30 can be positioned on a side oftest sensor 12.

Second end 28 includes a plurality of contact pads. In addition, secondend 28 can include a physical “key” such as a projection, protuberanceor notch 34 to require a unique orientation of test sensor 12 for theconnection or insertion of second end 28 into measurement module 14. Inother embodiments, second end 28 can include one or more electricalconnections to ensure the correct orientation and/or insertion of thetest sensor into the measurement module 14. Additionally, one or more ofthe electrical connections and contacts can be used to identify thespecific test sensor either by production lot for quality controlanalysis and/or identification of the type of test sensor, whichanalyte(s) the test sensor is configured to analyze and/or theanalyte(s) predicted concentration range.

Measurement module 14 includes a connection or receptacle 36 forreceiving second end 28. Receptacle 36 includes a corresponding “lock”for the physical key, if present, and a corresponding number ofelectrical contacts to mate with the electrical or magnetic connectorsand contacts on test sensor 12.

In one embodiment, measurement module 14 includes at least onebipotentiostat. The bipotentiostat can be configured to simultaneouslyapply and control the voltage of two different electrode sets on testsensor 12. In other embodiments, measurement module 14 can include twoor more bipotentiostats, each bipotentiostat configured to apply andcontrol the voltage of two different electrode sets. Consequently, themeasurement module can include one or more programmablebipotentiostat(s) to control potentials on the electrode structures incontact with the sample. In still yet other embodiments, thebipotentiostat can be included either in a desktop or hand-held meter 16described below.

Additionally, measurement module 14 can include hardware, software, orfirmware providing instructions to run one or more analyses andidentification of one or more selected analytes in a test sample.

Measurement module 14 also includes a connector 38 to operably couplemeasurement module 14 with portable controller 16. In the illustratedembodiment, measurement module 14 includes a connector 38, which isconfigured to be received within a receptacle 40 on portable controller16.

Portable controller 16 can be provided in a wide variety of hand-heldelectronic devices. In one preferred embodiment, portable controller 16is provided as one of a wide variety of Portable Digital Assistants(PDA), which are commercially available. In other embodiments, portablecontroller 16 can be provided as a portable (preferably dedicated)computer or CPU. Portable controller 16 includes a visual output screen46 and can, but is not required to include, one or more input devices44, buttons, switches, and the like. In addition, as is common with awide variety of commercially-available PDAs, data input/output screen 46can also allow input via a stylus 48.

In use, when measurement module 14 is operably connected to portablecontroller 16, a software program resident on measurement module 14 canbe automatically uploaded to portable controller 16. The uploadedprogram begins running on portable controller 16, prompting users toinput specific information and/or providing instructions to the users torun specified tests. In addition, the software can include one or moreinstructions or the capability for determining values for steady statecurrent, storing these values, calculating analyte concentrations, datamanagement, quality control, calibration, test sensor identification(production lot, analyte concentration range, and/or type of analyte),and connectivity to a centralized laboratory information system.

In one embodiment, controller 16 begins with a Drug Monitoring Systemscreen with date and time. The next screen prompts the user to enter theoperator identification. Thus, if desired, controller 16 could be set upso that only specified users with proper training could get access torun an assay. The operator identification can be entered as numbers oran alphanumeric code. The next command or next screen can be a main menuscreen that allows selection of a specific Drug Test, Control Test, orReview Results, for example. Selecting “Drug Test” prompts the user toenter a patient identification or name. The next screen then allows theuser to select the proper test(s) or conditions. Additionally controller16 (or measurement module 14) can include recognition software orhardware to verify and identify the specific test sensor that is orshould be used with either the patient or the selected test or testconditions. After selection of a specific test, the user is instructedto insert the test sensor or test sensors into the measurement module14. Controller 16 can block a test if the tests selected and/or if theinserted test sensor is not compatible or recognized. If the test sensoris compatible or recognized, then the controller is ready for the sampleto be supplied to the test sensor. Once the sample is applied, the testbegins. The controller can signal the user when the test is completedand report the results at the end of the assay period. In preferredembodiments, the controller has the capability to report quantitative orqualitative values depending on the desired requirements for an assay.Results are saved in the instrument and a report can be printed via theIR port 41 of the instrument directly to a printer equipped to receivean IR signal. The data can also be downloaded via an IR port, hardwireconnection and/or with a manual “hotsync” of the controller placed in acradle.

FIG. 2 is a perspective view of one embodiment of a test sensor 50 foruse with the present invention. Test sensor 50 is illustrated to analyzea plurality of different analytes in a single sample fluid. Test sensor50 includes a single dosing or sample port 51 and a plurality ofchannels 52 leading to a plurality of reagent chambers 53. Differentreagents, buffers, labeled ligand analogs, and the like are disposed ineach different reagent chamber 53 a, 53 b, 53 c . . . . It will beunderstood that two or more different reagent chambers, each containinga different reagent or sets of reagents, can be used for different assaymethods, e.g., sequential binding or displacement binding technique. Achannel 54 leads from the reagent chamber 53 to measurement zone 55.Again, a separate channel leads from each separate reagent chamber to adifferent detection chamber.

In the illustrated embodiment, soluble reagents, buffers, and/or labeledligand analogs are dried but not immobilized onto a substrate or matrix.A portion of the fluid sample is drawn into sample port 51 typically bycapillary action. The sample fluid progresses through channel 52 to eachof the reagent chambers, where the analytes in the sample bind to abinding partner, a labeled binding partner in a direct binding analysisor conversely the analyte can displace a bound partner from an analyte,a derivative thereof, or labeled ligand analog. The sample fluid withthe reaction product, from the reagent chamber, progresses to thedetection chamber, where the resulting labeled ligand analog conjugatecan be electrochemically detected.

In other embodiments, one or more of the reagents, buffers, and labeledligands can be immobilized either in the reagent chamber of in anotherportion of the fluid circuit on the test sensor; for example, in thedetection chamber.

FIG. 3 is perspective view of an alternative embodiment of a test sensor60 configured similar to test sensor 50, consequently the same referencenumbers are used for similar structures. Test sensor 60 differs fromsensor 50 by including separate dosing ports 61 a, 61 b, 61 c, . . . onefor each of the separate reagent chambers 63 a, 63 b, 63 c. In thisembodiment, different samples can be applied to the different ports 61a, 61 b, 61 c . . . and each of the different samples can be analyzedusing the same reagents and conditions. Alternatively, the same bulksample can be introduced to the different ports 61 a, 61 b, 61 c . . .and the different reagent chambers 63 a, 63 b, and 63 c can includedifferent reagents to perform different analysis of the bulk sample.

FIG. 4 is an exploded view of yet another embodiment of a test sensor 70for use with the present invention; FIG. 5 is a perspective view of testsensor 70. Test sensor 70, similar to test sensors 50 and 60, includes aplurality of reagent chambers and electrode structures. Test sensor 70includes a plurality of support strips 72, 74, 76, 78, and 80 laminatedon top of each other. In the illustrated embodiment, each supportincludes a sample port, reaction chamber, measurement zone and anelectrode structure. In one form, each of support strips 72, 74, 76, 78,and 80 includes a sample port 82, 84, 86, 88, and 90, respectively, thatallow the sample to be introduced into a single sample port, forexample, port 82. The introduced sample will flow and dose each assay oftest sensor 70. Except for the sample ports, each support strip 72, 74,76, 78, and 80 is separated from the adjacent test sensor by a layerthat is impervious to the sample and reagents. In addition, each of thereaction chambers of the sensor can include the same or differentreagents.

In use a sample is introduced into a single port such as port 82, wherethe sample flows to the remaining sample ports. The sample then flows toa reaction chamber and then to a measurement zone, where resultingspecies are interrogated. Test sensor 70 can be inserted into a meterthat is configured to receive a laminated test sensor with electrodepads vertically spaced apart to provide a visual display of the resultsof the test(s).

FIG. 6 illustrates one embodiment of a diagnostic kit 100 that includesa test sensor 102 and a meter 104. Test sensor 102 is inserted into themeter followed by dipping into the sample to the “dip line” 103. Testsensor 102 can be any of the test sensors described above.

FIG. 7 illustrates another embodiment of a diagnostic kit 110 configuredfor multi-analyte testing that can be dosed with a single sample, suchas, with blood from a finger pricked with a lancet. The single samplecan be analyzed for the presence and/or amount of many differentanalytes.

FIG. 8 illustrates a diagnostic kit 120 with a test sensor 122 and meter124 configured for single assay that can be dosed with a pipette.

FIG. 9 illustrates a diagnostic kit 130 that includes a test sensor 132and meter 134 configured for multi-analyte testing. Test sensor 132 isfixedly mounted on a wall of a sample collection chamber 136. Connector133 makes electrical connection to the contacts of test sensor 132. Inthe illustrated embodiment, the test sensor 132 is attached to the lid138 of a cup. This embodiment provides particular advantages by allowingthe collection of a sample. The collection chamber can then be sealedand stored or preserved as desired. Further, connection of connector 133opens a seal between the sample application port and the collectionchamber. The sample can be tested immediately upon collection or at alater time. In other embodiments, a test sensor can be mounted in or onanother wall of the collection chamber 136. In other embodiments thetest sensor 132 is removably mounted to the collection chamber 136.

FIG. 10 is a partial cross-sectional view of a microelectrode array 160according to the present invention illustrating the conditions ofsteady-state current. The partial microelectrode array 160 includes twocathodes or reducing electrodes 161 and 163 and an anode or oxidationelectrode 162. The mediator, M, is alternatively reduced at the cathodeelectrode 161 (or 163) and oxidized at the anode electrode 162. The gapbetween cathode electrode 161 and anode electrode 162 represented byreference line 166 can be selected to maintain a steady state currentand, consequently, allow for signal amplification as discussed morefully below. As noted above, the electrode structure includes areference electrode and at least first and second working electrodesdimensioned to allow diffusional recycling of the redox reversibleconjugate in the sample when a predetermined potential is applied to theworking electrodes. Smaller dimensions of finger widths W and gaps W_(g)increase the redox recycling, but increasing the length and number ofelectrode pairs is also desirable for effective current amplification.The gap represented by reference line 166 can be selected as desiredconsidering the analyte of interest and its concentration or predictedconcentration in the sample. Typically the gap between adjacentelectrodes is selected to be less than about 25 μm, preferably less thanabout 10 μm, more preferably less than about 2 μm. In cases where verylow sensitivities are needed, submicron gaps are desired.

FIG. 11 is a plan view of one embodiment of an Interdigitated Array(IDA) 170. IDA 170 is illustrated as a planar electrode structuresuitable for measuring redox recycling with a bipotentiostat inaccordance with this invention. IDA 170 includes two working electrodes172 and 174 (as drawn) defining six pairs of electrode fingers 176. Alsoincluded in IDA 170 are a reference electrode 178 and counter electrode180. The gap between two adjacent fingers represented by 166 of FIG. 10and the total number of fingers can be selected for IDA 170 as desiredfor a particular analyte of application. In preferred embodiments, it isdesirable to produce IDAs with more electrode pairs than shown in IDA170. For example, in order to achieve the proper amplification it ismost desirable to have at least 25 pairs of electrodes, more preferablyat least 50 pairs of electrodes or 750 electrode pairs, and even greaterthan 1000 pairs of electrodes. Amplification increases with decreasingthe width and gap and increasing the length and number of finger pairs.Each of the electrode structures in IDA 170 are dimensioned to allowdiffusional recycling of a diffusible redox reversible mediator in thesample when the electrodes 172 and 174 are poised at predeterminedanodic (oxidation) and cathodic (reduction) potentials.

Microelectrode arrays can be fabricated using a variety of technologiesincluding but not limited to photolithography, electron beamlithography, ion beam milling, nanoimprint lithography, and laserablation techniques described in WO 03/044511, which is incorporated byreference. Interdigitated electrode array (IDA) can be deposited on avariety of insulating substrates not limited to: glass, silicon, Upilex,Kapton, Kaladex, Melinex, or other polymeric substrates.

Improvements in meter construction and design for biosensor systems aredescribed in U.S. Pat. Nos. 4,999,632; 5,243,516; 5,366,609; 5,120,420;5,141,868; 5,192,415; 5,264,103; 5,352,351; 5,405,511; 5,437,999;5,438,271; and 5,575,895, the disclosures of which are herebyincorporated by reference.

The size (or surface area) and number of electrode pairs can be selecteddepending upon the analyte(s), their concentration, and the samplemedium among other factors. In addition, the present invention providesan empirical construct for selecting the size and/or number of pairs ofelectrode fingers for a given set of conditions. The construct isdescribed more fully below.

In yet other embodiments, the components of array 170 can be sized toprovide a macro electrode array. The electrode dimensions and the gapbetween the electrodes for the macro array can vary significantly andmay be limited only by the size of the test sensor and the availablesample volume.

Vertical IDA Electrodes

FIG. 12 is a side or edge view of a vertical interdigitated array (VIDA)190 in accordance with the present invention. FIG. 13 is a top plan viewof one embodiment of array 190. Array 190 includes a base or insulativesubstrate 192 onto which is deposited an electrically first conductivematerial as conductive layer 194 to provide a first electrode 195. Adielectric insulative layer 196 is deposited on conductive layer 194 andsubstrate 192. A second conductive material 193 is deposited on top ofthe dielectric layer 196. A second dielectric layer (not shown) is thendeposited and patterned onto the second conductive material 193 todefine a plurality of non-conductive fingers (not shown). The exposedsecond conductive material is removed followed by the removal of theexposed dielectric layer. This leaves behind the second set of electrodefingers 193 deposited on top of the non-conductive finger 196 andpatterned to define a plurality of electrode fingers 198.

The electrode gap of a VIDA is defined by the thickness of the dielecticinsulator 196 sandwiched between the conductive layers, thus micron tosubmicron gaps can be produced using standard techniques that are notcapable of submicron resolution. This can be achieved because the VIDAfinger gap or feature size is not dependent upon the limits of thepatterning techniques but is rather a function of how thin (or thick)the dielectric insulator that can be applied.

In one embodiment, the gap is selected to be less than about 1 μm. Inother embodiments, the desired gap width is selected to be between 1 μmand 3 μM.

Sidewalls 202 and 204 and an upper plate or roof 206 can be fabricatedover the vertical electrode array to define a detection chamber 208. Thetotal volume of detection chamber 208 can be selected as desired andonly limited by the desired size of the test sensor and the number ofdetection chambers formed on the strip.

The VIDA 190 provides additional advantages over those provided by theIDAs described above, including a higher density of electrode pairs perunit surface area. Consequently, smaller chambers can be fabricated thatcontain the same number of electrode pairs, each electrode having thesame surface area as the planar IDA. Furthermore, a test sensor caninclude a larger number of the VIDA as a test sensor that includes onlyIDAs, again where the surface area of the two test sensors are the sameand the chambers each contain the same number and sized electrode pairs.This can provide important improvements for test sensors, which areconfigured to simultaneously detect/analyze a number of differentanalytes.

In FIG. 12 illustrating a VIDA electrode, the dielectric layer 196 isfree of holes in the regions between the two working electrodes. TypicalVIDA designs involve the fabrication of a metal-dielectric-metal on arigid or flexible substrate. The integrity of the dielectric layer iscritical for the electrochemical function of the device. Breakdown ofthe inner dielectric will lead to electrical shorts and a nonfunctionaldevice. Insulators can be deposited by various processes includingsputtering and spin coated dielectrics. A dry etching technique can beused to remove the insulator layer in the desired regions down to thefirst conductive layer. The process of making VIDA electrodes is notlimited the aforementioned procedure that was included only as anexample.

Cell Constants

Solution resistivity is an intrinsic property of a solution determinedby the combined concentrations and mobilities of all dissolved ions in asystem. This resistivity (ρ) in Ohm×cm of a sample will be influencedthe sample matrix and reagents that mix the sample includingelectrochemical labels, buffers, salts, and antibody to name a fewexamples.

When an electric field is applied between two electrodes in the cell,ions move until the double-layer at the electrode interfaces chargessufficiently to oppose and eventually cancel the applied field. Thedouble-layer charging behaves like a capacitor in series with thesolution resistance. The measured solution resistance will have aconstant additional series resistance due to contact resistance andintrinsic electrode material resistance. High frequency resistancemeasured in an electrochemical cell is proportional to the intrinsicsolution resistivity p and the proportionality constant is called the“cell constant”. High frequency may be defined as a range of frequencieswhere the electrochemical cell's reactive (capacitive) properties may besafely ignored.

The cell constant is an important factor in the sensitivity of the cellto changes in solution resistivity, and varies widely with electrodeconfiguration. The geometry of interest to this invention is aninterdigitated array (IDA). Here, a number of coplanar anode and cathodepairs are repeatedly alternately interlaced or interdigitated to form alarger interdigitated array electrode. For an interdigitated array, thehigh frequency resistance is related to the solution's resistivity byEquation 1 below.

$\begin{matrix}{R = \frac{\rho}{mbG}} & (1)\end{matrix}$

The cell constant for an IDA is therefore defined by Equation 2, where mis the number of microband electrode pairs of the IDA, b is the lengthof the bands in cm, and G is a dimensionless function of the electrodegeometry finger width (W) and gap (W_(g)). G can be approximated as perthe expression defined by Equation 3. The cell constant is a usefulparameter of an electrode configuration that can be both calculated andmeasured. The cell constant of an IDA electrochemical cell depends onits architecture, especially the electrode geometry, although in somecases the top boundary of the cell (capillary height) can also play arole. Estimation of the cell constant for an IDA is derived from theequations and work of Aoki and others, but is only a function of thenumber of IDA electrode pairs and their dimensions. Therefore, the cellconstant is a single value that can be used to compare various electrodegeometries. Electrodes with the same cell constants will have similarcharacteristics in the application of electrodes of this invention.

$\begin{matrix}{{{Cell}\mspace{14mu} {{Const}.}} = \frac{1}{mbG}} & (2)\end{matrix}$

G may be estimated for an IDA from the approximation Equation 3 fromAoki.

$\begin{matrix}{G = \left( {{\frac{2}{\pi}{\ln \left( {2.55\left( {1 + \frac{W}{W_{g}}} \right)} \right)}} - {0.19\left( \frac{W_{g}}{W + W_{g}} \right)^{2}}} \right)} & (3)\end{matrix}$

where Wg is the width of the gap between adjacent working andcounter-electrode bands and W is the width of each (electrode finger)microband. The model approximation assumes no array-edge effects,meaning the electrochemical cell, containing the array and electrolyte,is large enough so as not to distort electric field lines at the cellboundaries (i.e., IDA must have enough fingers to prevent the lack of aneighbor for the first and last finger from significantly altering thecurrent). The model also assumes that the microband anode and cathodeelectrode fingers are the same width. As W_(g)/(W+W_(g)) varies from 0.1to 0.9, G varies from about 2.5 to about 0.5. When W=W_(g),W_(g)/(W+W_(g))=0.5 and G is about 1.

Many articles have appeared in the literature from Koichi Aoki andothers to better understand, model and predict responses atinterdigitated array electrodes. The following publications are a sampleof many of the discussions and mathematical computations that have beenpublished in the field: “Theory of chronoamperometric curves atmicroband electrodes”, J. Electroanal. Chem., 225 (1987) 19-32;“Derivation of an approximate equation for chronoamperometric curves atmicroband electrodes and its experimental verification”, J. Electroanal.Chem. 230 (1987) 61-67; “Quantitative analysis of reversiblediffusion-controlled currents of redox soluble species at interdigitatedarray electrodes under steady-state conditions”, J. Electroanal. Chem.,256 (1988) 269-282; “Time-dependence of diffusion-controlled currents ofa soluble redox couple at interdigitated microarray” J. Electroanal.Chem., (1989) 11-20; and “Approximate models of interdigitated arrayelectrodes for evaluating steady-state currents”, J. Electroanal. Chem.284 (1990) 35-42.

Equation 4 was also derived by the aforementioned literature and is usedto evaluate reversible diffusion-controlled currents of redox solublemediators on IDA electrodes under steady-state conditions. This equationwas used to predict the slope for the IDA and VIDA electrodes of FIG.15. In this equation, m is the number of microband electrode pairs ofthe IDA, b is the length of the band in cm, n is the number of electronsinvolved in the redox reaction, F is Faraday's constant[9.65E+04C/equiv], c is the bulk concentration of the redox molecule, Dthe diffusion constant of the redox molecule [For osmium free mediator,5E-06 cm²/sec.], W is the width of the microband electrodes (anode orcathode) in cm where W_(a)=W_(c), and Wg is the gap between respectiveanode and cathode electrodes.

$\begin{matrix}{{I} = {{mbnFcD}\left( {{\frac{2}{\pi}{\ln \left( {2.55\left( {1 + \frac{W}{W_{g}}} \right)} \right)}} - {0.19\left( \frac{W_{g}}{W + W_{g}} \right)^{2}}} \right)}} & (4)\end{matrix}$

One will note that there is a relationship between this equation topredict steady-state current, G, and the cell constant. The expressionfor the steady-state current can be rewritten as |I|=mbnFcDG.Rearrangement of the expression leads to the following relationship withcell constant (Equation 5 below). This implies that the steady state DCcurrent is inversely proportional to an IDA electrode's cell constant.Smaller cell constants should provide a proportionately higher steadystate DC current for a given concentration and diffusion coefficient.Smaller cell constants also produce faster transitions to steady stateDC conditions.

$\begin{matrix}{{{Cell}\mspace{14mu} {{Const}.}} = {\frac{1}{mbG} = \frac{nFcD}{I}}} & (5)\end{matrix}$

Equation 4 for the steady state current may be normalized to a unit areaby dividing the predicted current by the IDA electrode area ofinterdigitation (Area=2(W+W_(g))mb100). Equation 6 shows the equationused to calculate normalized predicted slope in FIG. 15. The current ismultiplied by 10⁹ to convert to nA and the area is multiplied by 100 toconvert from cm² to mm².

$\begin{matrix}{\frac{I}{Area} = {\frac{{mbnFDc}\; 10^{9}}{2\left( {W + W_{g}} \right){mb}\; 100}\left( {{\frac{2}{\pi}{\ln \left( {2.55\left( {1 + \frac{W}{W_{g}}} \right)} \right)}} - {0.19\left( \frac{W_{g}}{W + W_{g}} \right)^{2}}} \right)}} & (6)\end{matrix}$

FIG. 15 provides Table 3 listing of various embodiments of IDAs andVIDAs prepared. The electrode geometry for each electrode is evaluated.Equations 4 and 6 discussed above were used to model the specificelectrodes in terms of predicted and normalized predicted slope. Inaddition, experimental values are shown from recycling CVs andamperometric bipotentiostat dose response curves performed with either aCHI 802A or CHI 832A bipotentiostat from CHI Instruments, Austin, Tex.Table 4 shows the cell constant for four of these electrodes, but usingEquation 2 the cell constant for each electrode shown in FIG. 15 can becalculated including the VIDA electrodes.

In addition to using the equations to compare the predicted vs.experimental values, Equations 2, 4, and 6 were also used as a tool todesign electrodes of proper dimensions in order to achieve electrodesthat will achieve the desired response for specific immunoassays. Inorder to use the equations as a design tool, it was assumed that about0.5-1 nA would be the low end detection sensitivity for a massproduction bipotentiostat instrument. A prototype module meeting thesecriteria was designed and built to interface with a Handsprings VisorPDA. The planar IDA having 2 μm finger width and gap with 750 microbandpairs each 6 mm in length listed in Table 3 of FIG. 15 was designed andconstructed to improve the detection sensitivities to about 5 nM in theideal case (1000 nM/215 nA=4.65 nM/nA). This 2 μm IDA design achievedsignificantly improved amplification compared to the electrodes with 10,15, and 21 μm gaps as shown in FIG. 16. Assuming a 1 nA resolution andthe measured slope of 152 nA/μM, the sensitivity would be about 7 nM.

FIG. 49 is a graph obtained from an osmium biotin assay prepared usingthe aforementioned 750 finger, 2 μm IDA electrode. The assay wasconfigured for analyte measurement up to about 1000 nM based on theconcentration of osmium biotin. The lowest level tested with this assaywas 250 nM which was easily distinguished. Extrapolating the sensitivityto a 1 nA resolution provides an assay with a sensitivity of about 20nM.

The three-dimensional plot shown in FIG. 14 shows normalized current vs.electrode dimensions for a series of planar IDAs of varying W and W_(g).FIG. 14 shows that even with μm spaced IDAs, significant improvements innormalized currents can be made by decreasing W and W_(g)

FIG. 16 is a graph plotting the current (in nA) measured on differentIDA electrodes using the osmium free mediator (Bis(2,2′-Bipyridyl)Imidazole Chloro Osmium (III)dichloride). The IDAs wereprepared as described herein and included electrode structures with 50finger pairs each having a width (W) of about 21 μm and a gap (W_(g)) of10, 15, and 21 μm. Another electrode structure had 750 finger pairs eachhaving W=W_(g)=2 μm. All electrodes had a finger length (b) of 6 mm.This plot illustrates the magnitude of IDA amplification that weretested on different IDA electrode configurations. One feature of the IDAelectrodes of the present invention is that rather large electrode areas(36 mm²) and large number of fingers (750 pairs) can be fabricated andused for electrochemical immunoassays. From the graphs and equations itwas observed that decreasing W and W_(g) and increasing the number offingers all can contribute to an increase in the measurable current onan IDA. FIG. 16 also illustrates the particularly small current obtainedfor an IDA when there is no redox recycling.

The “Cell Constant” is a useful value that can be both calculated andmeasured for a particular electrode configuration. The characteristicsof two electrodes of the same cell constants should function similarlyin the application of electrodes of this invention and can be used tocompare various electrode configurations. Electrodes of interest forthis invention are electrodes with about equal or smaller cell constantsthan those used to perform the homogeneous electrochemical immunoassaysof this invention as shown in Table 4. Preferably the IDA cell constantfor electrodes of the present invention is less than about 0.03 cm⁻¹ foranalytes at high concentrations such as theophylline, even morepreferred would be electrode structures with a cell constant of lessthan about 0.02 cm⁻¹, and most preferably smaller than about 0.002 cm⁻¹.IDA electrodes whose cell constants are less than about 0.0025 cm⁻¹provide good redox recycling and amplification with the osmiumelectrochemical mediator conjugates discussed herein for homogeneouselectrochemical immunoassays. Table 4 below lists the cell constants forelectrodes of this invention along with the cell constants for some ofthe typical IDA electrodes that have been reported and studied forvarious electrochemical measurements. It is apparent from the table thatthe electrodes that are preferred for this invention have smaller cellconstants than those commonly described in the literature although notnecessarily smaller electrode gaps and widths. Although most researchersare moving towards more closely spaced IDA electrode widths and gaps,they have also often significantly reduced the finger length and thusthe electrode cells are much smaller in overall dimensions. Thus, thesmall cell constants desired for this invention has usually not beenrealized.

Techniques such as E-beam lithography are becoming more commonly used toproduce IDA electrodes with small features. At the present time, thistechnique is not amenable to high volume, low cost disposable sensors.The preferred electrodes of this invention are designed to achieve therequired signal amplification based on the required immunoassaysensitivity. Electrodes that are amenable to high speed reel to reelprocessing such as standard photolithography techniques and laserablation are most preferred for disposable immunoassay sensors. Designsthat minimize the cell constants, not necessarily the smallest electrodefeatures, are the preferred electrode designs. Small electrode featuresare desirable if the cell constants are also decreased which can beachieved by adjusting the length and/or number of IDA fingers.

TABLE 4 Electrode structures of invention or m = pairs of b = lengthCell cited in microband W = Electrode W_(g) = Electrode of band Constantliterature. electrodes width (μm) gap (μm) (cm) (cm⁻¹) Immunosensor 7502 2 0.6 0.0022 electrodes per 150 5 5 0.3 0.0215 this invention 50 21 100.6 0.0254 50 21 15 0.6 0.0290 50 21 21 0.6 0.0323 J. Electroanal. 25 105 0.2 0.1546 Chem., 256 50 5 5 0.2 0.0970 (1988) 269-282 50 3 5 0.20.1132 25 10 2 0.2 0.1152 50 5 2 0.2 0.0718 50 3 2 0.2 0.0850 Analytica70 3 1 0.09 0.1074 Chimica Acta 35 1.5 0.7 0.09 0.2399 305 (1995) 35 1.50.5 0.09 0.2148 126-136 35 1.5 0.3 0.09 0.1828 224 1 0.5 0.09 0.0383

Enzyme Amplification

As an alternative to IDA amplification, homogeneous electrochemicalimmunoassays can also be developed with enzyme amplification. For someantigens, the enzyme amplification was the preferred method since redoxrecycling of the prepared mediator did not recycle properly with theconjugate. One example was with an osmium morphine conjugate shownbelow.

Several examples of enzyme assays were developed including: cocaine,morphine, THC, and biotin. The premise for enzyme amplification is thatthe electrochemical mediator label is reduced by the enzyme and oxidizedon the electrode surface. Since enzymes are very efficient, this methodcompetes well with IDA amplification techniques and is applicable tomany assays. There are, however, several disadvantages with enzymeamplification including the ability to amplify sources of noise such asinterfering substances. Addition of enzyme to the device also requiresadditional reagents to ensure enzyme stability. In addition, one mustalso balance the preferred pH of the enzyme vs. antibody. Advantagesinclude simpler electrode structures similar to those used in glucosesensors.

Assay Schemes

Various assay schemes of this invention are described below. All ofthese schemes require that the reagents are dried in one or more regionsof the test sensor. In order for the assays to be viable, fastsolubility of the reagents are required with the addition of sample. Forthis reason, it is highly desirable that the electrochemical labels,binding partners, and other supporting reagents solubilize in the samplematrix. For this reason, conjugates of hydrophobic antigens will oftenrequire the use of hydrophilic linkers to improve the solubility of thereagent such as the osmium-PEG-THC-2 (compound 37) and PEG-methotrexate(compound 38). In addition, the hydrophobic nature of the antigen itselfin the sample and on the conjugate is also a concern for nonspecificbinding and hydrophobic interactions which can lead to a diminishedconjugate response and assay result due to less antigen available forbinding. Conjugates of hydrophobic antigens may also aggregate or formmicelles since they are hydrophilic on one end and hydrophobic on theother. Electrode fouling or contamination which inhibits theelectrochemistry is also a concern. The use ofhydroxypropyl-β-cyclodextrin added to the reagents has been shown togreatly improve assays with these concerns possibly allowing thehydrophobic portion to go inside the cavity of cyclodextrin. It has alsobeen shown that the use of the hydroxypropyl-β-cyclodextrin does notappear to inhibit the binding events.

Several assay schemes can be used in accordance with the presentinvention. The schemes include both homogeneous and heterogeneousimmunoassay methods. The preferred method is the homogeneous competitiveimmunoassay that enables the direct measurement of unbound mediatorconjugates without separation from the bound mediator conjugate. Thusthe immunoassay can be carried out in one reaction mixture.Heterogeneous immunoassays, which will not be discussed in detail,require the use of a separation step to separate bound mediatorconjugates from the free labeled.

Homogeneous immunoassays can be more readily adapted to a “point ofcare” or “in field” device of this invention. Two types of competitivebinding assays have been employed. The first competitive binding assayis a displacement assay where the mediator labeled antigen is pre-boundto the antibody (or other binding partner). Then with the addition ofantigen under non equilibrium conditions, the mediator labeled antigenis displaced. The assay can then measure the amount of unbound mediatorlabel and correlate it to the concentration of the analyte. The secondcompetitive binding assay is one in which the antigen is first broughtin contact with excess antibody (or other binding partner) followed bythe addition of the mediator labeled antigen which binds to theremaining antibody (or other binding partner). As with the displacementassay, the assay can then measure the amount of unbound mediator labeland correlate it to the concentration of the analyte.

The specific activity of the electrochemical mediator labeled antigen ismodulated according to the analyte concentration in the sample. Theresulting current will be a function of the amount of analyte present.

FIG. 17 illustrates one embodiment of a competitive electrochemicalhomogeneous immunoassay test sensor that uses sequential binding. Thefigure illustrates a capillary test sensor where different reagents andthe electrode structure are located. In this embodiment, the reagents(antibody and conjugate) are dried in a soluble matrix in locationsupstream of the measurement zone. The measurement zone also includes theactive electrode areas. The sample which may contain antigen is appliedto a sample application port in sufficient volume to fill the capillary.As the sample fills the capillary and dissolves the reagents, thevarious binding events take place. When the sample and reagents reachthe measurement zone, unbound mediator conjugate can be measured.Several variations of this basic system can be envisioned and includedas part of this invention. In another embodiment, the reagents are mixedtogether and dried in the measurement zone either on the activeelectrode surface or on another surface of the measurement zone. In thisconfiguration, no additional chambers or regions are required.

The illustrated homogeneous electrochemical immunoassay is based onspecific affinity between antigens and antibodies. The antigen ofinterest (e.g. drug, peptide, or biotin) is labeled with anelectroactive redox mediator to yield the redox reversible mediatorlabeled antigen (mediator conjugate). The sample being assayed is mixedwith a predicted excess of the antibody to the antigen of interest. Ifthe antigen is present in the sample, binding between the antigen andthe antibody occurs. The resulting mixture of bound antigen-antibody andthe excess unbound antibody is then combined with the redox reversiblemediator labeled antigen. The unbound antibody binds to the antigen ofthe redox reversible conjugates to form a bound complex. The resultingmixture contains the redox reversible unbound conjugate, and the boundconjugate. The redox recycling of the bound conjugate is inhibited bythe binding of the large molecular weight binding partner. In thisembodiment, a predetermined amount of the specific antibody to theantigen of interest is combined with the sample, and thereafter, isadded to a predetermined amount of the redox reversible conjugate.Applying a potential selected to induce the unbound redox reversiblelabel to undergo redox recycling at the electrodes generates a current,which can be measured and correlated with analyte concentration.

The above illustration is based on specific affinity between antigensand antibodies. It will be understood that any analyte of interest andits specific binding partner can be used in place of theantigen/antibody combination.

In alternative embodiments, it is also possible to combine thepredetermined amounts of the antibody with the redox reversibleconjugate to form the respective complexes prior to combining thosecomponents with the liquid sample. In the latter case, the redoxreversible conjugate will be displaced from its respective antibody bythe corresponding antigen to provide a concentration of the redoxreversible conjugates proportionate to the concentration of antigen inthe liquid sample.

The reagents, that is, the predetermined amounts of the antibody for theantigen and the predetermined amounts of the corresponding redoxreversible conjugate can, for example, be deposited in a vessel forreceiving a predetermined volume of the liquid sample. The liquid sampleis added to the vessel, and thereafter, or simultaneously, the liquidsample is contacted with the electrode structure.

Two homogeneous immunoassay formats can be implemented for assays:displacement and, sequential binding. Both assays electrochemicallydetect the amount of free (unbound) conjugate at the end of the assaysequence. Interdigitated array (IDA) electrodes or enzymes are used toamplify the current signal through redox cycling of the mediator label.Measured current due to redox cycling is proportional to the amount offree (unbound) conjugate and increases with analyte concentration. Inthe case of IDA amplification, a steady-state response is obtainedwithin seconds of applying the sample and the proper redox potentials tothe first and second working electrodes via a bipotentiostat.

Recycling with a bipotentiostat should result in two measurements thatare equal in magnitude but opposite in sign. This provides distinctadvantages in determining if there is an interferent effect due to a nonrecycling interferent which would cause deviation from the expectedvalue. Typically when interferents are present in low enoughconcentrations and when they do not undergo redox recycling, thepotential interferents are not present in the final steady state currentafter a short period of time. Non recycling interferents atconcentration of 10 to 100 times the concentration of the desiredanalyte can still be negligible when using redox recycling measurementssince the interferent does not recycle. Interferents at significantlyhigher concentration, i.e. as is possible with ascorbic acid in urine,can show a large increase in the oxidation response and a smallerdecrease in the reduction response from the expected result. It isexpected that by use of both the oxidation and reduction response, theanalyte of interest may be able to be corrected by mathematicalcomputations.

Ascorbic acid (Vitamin C) concentrations in biological samples can varydramatically. In random urine samples, the concentration can rangebetween 60-400 μM. In plasma the range is 34-91 μM and in whole blood40-114 μM. Ascorbic acid is a strong reducing agent, thus it is readilyoxidized and can be a source of electrochemical interference withclinical tests.

In one study, the effect of ascorbic acid was studied on 21 μm IDAelectrodes using osmium free mediator at 1, 5, and 10 μM. The ascorbicacid was varied at 100, 200, and 400 μM. Measurements were made using aCHI 832A bipotentiostat with WE1=250 mV, WE2=0 mV. The 100 μM sampleshowed little deviation from the control but the 200 and 400 μM severelyshows the reductive properties of ascorbic acid. In comparison to thecontrol, the 200 and 400 μM samples, the oxidation currents were greatlyincreased and the reduction currents were diminished. For oxidation, anincrease in the slope of the OSFM response was observed as well as anincrease in the Y intercept. For reduction the opposite trend wasobserved, a decrease in the slope and decrease in the Y intercept. It isexpected that faster recycling IDA electrodes (electrodes with smaller Wand W_(g)) would have less interferences from non redox recyclinginterferents.

Sample Treatment

The method can be performed on liquid samples comprising biologicalfluids such as saliva, urine, or blood, or the liquid sample can bederived from environmental sources. The liquid samples can be analyzedneat “as is” or pre-processed such as diluted with a buffered solution,concentrated or otherwise processed to optimize detection of thetargeted analyte(s). Thus, for example, blood samples can be lysedand/or otherwise denatured to solubilize cellular components. In anotherexample urine samples can be mixed with a predetermined amount ofascorbate oxidase. Hydroxypropyl-β-cyclodextrin may also be added tosamples containing an analyte of hydrophobic nature to minimizenonspecific binding of the analyte to the surface of the samplecollection containers, reagent, or measurement zones. The method can beperformed using widely variant sample handling techniques.

The present invention includes at least one electrochemical label foreach analyte to be detected and/or analyzed. Illustrative examples of anosmium electrochemical label for use in this invention are complexes ofthe Compound 1:

wherein R and R₁ are the same or different and each can be selectedfrom: 2,2′-bipyridyl, 4,4′-disubstituted-2,2′-bipyridyl,5-5′-disubstituted-2,2′-bipyridyl, 1,10-phenanthrolinyl,4,7-disubstituted-1,10-phenanthrolinyl, or5,6-disubstituted-1,10-phenanthrolinyl, wherein each substituent is amethyl, ethyl, or phenyl group, and where the R and R₁ groups arecoordinated to Os through their nitrogen atoms; q is 1 or 0; R₇ isB-(L)_(k)-Q(CH₂)_(i); R₂ is hydrogen, methyl, or ethyl when q is 1, andR₂ is B-(L)_(k)-Q(CH₂)_(i)—when q is 0; for the groupB-(L)_(k)-Q(CH₂)_(i): Q is O, S, or NR₄, wherein R₄ is hydrogen, methylor ethyl; -L—is a divalent linker; k is the integer 1 or 0; i is aninteger 1, 2, 3, 4, 5 or 6; and B a group comprising a ligand capable ofbinding to a specific analyte binding partner; Z is chloro or bromo; mis +1 or +2; X is counter ion such as a mono- or divalent anion, e.g.,chloride, bromide, iodide, fluoride, tetrafluoroborate, perchlorate,nitrate, sulfate, carbonate, or sulfite; and n is selected to provide aneutral salt.

A second illustrative example of an osmium electrochemical label for usewith the present invention is represented by Compound 2:

wherein R and R¹ are the same or different and each can be selectedfrom: 2,2′-bipyridyl, 4,4′-disubstituted-2,2′-bipyridyl,5-5′-disubstituted-2,2′-bipyridyl, 1,10-phenanthrolinyl,4,7-disubstituted-1,10-phenanthrolinyl,5,6-disubstituted-1,10-phenanthrolinyl, or N,N′-dimethyl2,2′-biimidazole, wherein each substituent is a methyl, ethyl, or phenylgroup, and where the R and R¹ groups are coordinated to osmium throughtheir nitrogen atoms; R² is hydrogen, methyl, or ethyl; -L—is a linker;E is a trivalent linker; B is a group comprising a ligand capable ofbinding to a specific analyte binding partner; Z is chloro or bromo; Xis a counter ion; and y is selected to provide a neutral salt; and m is2 to 4.

A third illustrative example of an osmium electrochemical label for usewith the present invention is represented by Compound 3.

wherein, R and R¹ are the same or different and each can be selectedfrom: 2,2′-bipyridyl, 4,4′-disubstituted-2,2′-bipyridyl,5-5′-disubstituted-2,2′-bipyridyl, 1,10-phenanthrolinyl,4,7-disubstituted-1,10-phenanthrolinyl,5,6-disubstituted-1,10-phenanthrolinyl, or N,N′-dimethyl2,2′-biimidazole, wherein each substituent is a methyl, ethyl, or phenylgroup, and where the R and R¹ groups are coordinated to Os through theirnitrogen atoms, R² is a saturated or unsaturated, substituted orunsubstituted, straight or branched chain, hydrocarbyl group having 1-10carbon atoms; —R³ is H, CH₃ or C₂H₅; L is (CH₂)_(i)Q wherein i is aninteger between 1 and 10, and Q is O, S, or NR³; B is a group comprisinga ligand capable of binding to a specific analyte binding partner; X isa counter ion; y is selected to provide a neutral salt; and m is from4-8.

A fourth illustrative example of an osmium electrochemical label for usewith the present invention is represented by Compound 4 below.

wherein, R and R₁ are the same or different and each can be selectedfrom: 2,2′-bipyridyl, 4,4′-disubstituted-2,2′-bipyridyl,5-5′-disubstituted-2,2′-bipyridyl, 1,10-phenanthrolinyl,4,7-disubstituted-1,10-phenanthrolinyl,5,6-disubstituted-1,10-phenanthrolinyl, or N,N′-dimethyl2-2′-biimidazole wherein each substituent is a methyl, ethyl, or phenylgroup, and where the R and R¹ groups are coordinated to Os through theirnitrogen atoms; R³ is H, CH₃ or C₂H₅; L is (CH₂)_(i)Q, wherein i is aninteger 1-10 and Q is O or NR³; B is a group comprising a ligand capableof binding to a specific analyte binding partner; Z is chlorine orbromine; X is a counter ion; y is selected to provide a neutral salt;and m is 1 or 2.

The precursor to compound 4 (where the B substituent is replaced with A:—(CH₂)_(J)—NR³, —(CH₂)_(j)—SH, or an activated ester wherein j is aninteger between 1-5) is also included within the scope of the presentinvention.

As illustrated above for Compounds 1, 2, and 3 the osmium mediatorconjugate(s) can be envisioned to comprise at least two and optionallythree components. The osmium mediator conjugate can include one or moreorganometallic osmium group(s), one or more linking groups, and a ligandanalog. The organometallic osmium species by itself, either as a freesalt or with a neutral imidazole group, is electrochemically detectable.The di-chelating ligand on the osmium center, i.e., the bipyridyl andphenanthrolinyl ligands, can be varied as desired to provide a conjugatehaving the desired properties including redox potential and solubility.For example, for some fluid samples or specific analytes it may bedesirable to employ an osmium conjugate that exhibits increasedhydrophobicity. Varying the di-chelating ligands from a 2,2′-bipyridylligand to a 1,10-phenanthrolinyl ligand increases the hydrophobicity ofthe osmium complex; similarly, adding alkyl substituents to thedi-chelating ligand also increases the hydrophobicity of the osmiumcomplex.

The linking group L for use in the present invention can be selecteddepending upon a variety of factors including the particular analyte tobe analyzed, its concentration, and the sample medium. In preferredembodiments, the linking groups can be selected based upon theparticular analyte(s) of interest, its (or their) concentration in thesample medium, and the sample medium itself. The linking groups of thepresent invention can also be divalent linking groups. In one form, thelinking group of the present invention can be selected to behydrophilic. It has been determined that appropriate selection of thelinking group can greatly influence the detection and analysis ofparticular analytes. For example, highly hydrophilic analytes caninfluence the mobility of the redox reversible conjugate in the samplemedium in the reaction chamber. This, in turn, can affect the currentamplification via either diffusional recycling under steady stateconditions and/or enzyme recycling.

There are many types of conjugation chemistry that can be employed tolink the osmium mediator to a ligand analog. The following conjugationchemistries employed for the preparation of osmium mediator-peptideconjugates have also been commonly used for preparing proteinconjugates: 1) formation of amide bond by reactive ester with primaryamine; and 2) formation of thioether bond by maleimide with sulfhydrylgroup; and formation of a urea or thio urea linkage by reaction of anamino group of osmium mediator with an isocyanate or isothocyanatefunctionality of the drug derivatives Amide bond is preferred overthioether bond because amide bond is generally more stable. Based on thepreferred conjugation chemistry, the ligand on the osmium mediator canbe functionalized with either a primary amine group or a carboxylic acidgroup. The best location for these functional groups is believed to bethe C-4 or C-5 positions on the imidazole ligand of the osmium mediator;however, functionalization through the non-osmium-complexed imidazolering nitrogen atom can also be carried out.

In some osmium mediators, the amine group on the histamine ligand can bedirectly attached to the ligand analog, if a suitable reactive groupexists or can be provided on the ligand analog. For example, the aminegroup on histamine ligand of osmium mediator readily reacts with anactivated carboxyl group on methotrexate.

In one preferred embodiment of the present invention, one or more of thedivalent linking groups is selected to exhibit sufficient hydrophilicityto enhance the mobility of the redox recycling conjugate in an aqueousmedium. Examples of di-valent linking groups include: polyethyleneglycol PEG either as a monomer, dimer, oligomer or short chain polymer.

In alternative embodiments, the linker itself can be connected to one ormore crosslinking groups. For example, in the species illustrated abovefor either Compound 1, 2, or 3 a histadyl group (derived from histamine)is first attached to the osmium metal center. The histamine itself is afirst linking group or moiety. It will be understood that a wide varietyof linking groups can be used with the osmium complex. The resultingcomplexes are considered to be included within the scope of the presentinvention.

For the purpose of promoting further understanding and appreciation ofthe present invention and its advantages, the following examples areprovided. It will be understood, however, that these examples areillustrative and not limiting in any fashion.

EXAMPLES

The term “Osmium Free Mediator” or “Free Mediator” or “OSFM” all referto the use of bis(2,2′-bipyridyl)imidazole chloro osmium(III)dichloride, which is described in U.S. Pat. No. 5,589,326. Thismediator is often used as a model electrochemical mediator to evaluateand compare redox amplification on various IDA electrodes. The structureof this mediator is shown below.

The notation “PBS” refers to a Phosphate Buffer Saline matrix consistingof 10 mM Potassium Phosphate Buffer, 2.7 mM Potassium Chloride, and 137mM Sodium Chloride prepared from Sigma product #P4417 or a similarsaline buffer matrix.

The notation “PBST” refers to a Phosphate Buffer Saline matrixconsisting of 10 mM Potassium Phosphate Buffer, 2.7 mM PotassiumChloride, 137 mM Sodium Chloride, and about 0.1% to 0.5% Tween 20.

Reference to an “external Ag/AgCl” refers to a commercially availableAg/AgCl electrode such as the RE 803 mini-reference electrode fromAbtech Scientific Inc., Richmond, Va.

The term “internal Ag/AgCl” refers to a Ag/AgCl ink applied to thesample contact region of a one of the electrodes of the electrode cell.In most cases this was a commercially available ink (product E2414) fromErcon, Wareham, Mass. The internal reference electrode as constructed onour electrodes had a potential shift in comparisons to the externalAg/AgCl reference electrode by about 100 mV.

Preparation of Osmium Electrochemical Labels

The bis(2,2′-bipyridyl)imidazolyl chloro osmium (III)dichloride (OsmiumFree Mediator) has been shown to be an excellent electron mediator formany oxidoreductase enzymes (U.S. Pat. No. 5,589,326). It has fastmediation kinetics (about 500 times faster than ferricyanide withglucose oxidase) and a relatively low redox potential (+150 mV vs.Ag/AgCl). It has also a very fast electron transfer rate at electrodesurfaces. Importantly, the organic ligands on the osmium conjugate canbe functionalized so that it can be covalently linked to other moleculeswithout detrimental effects on redox properties of the osmium center.These unique properties of osmium conjugate make it an idealelectrochemical label for affinity sensors. The osmium mediators can beprepared according to the procedure described in U.S. Pat. Nos.6,294,062; 6,352,824; and 6,262,264, which are incorporated by referencein their entirety herein.

Osmium mediators with these new ligands were synthesized using the sameor similar procedure used to synthesize the osmium free mediator. Theirsynthesis consists of two major process steps as outlined below. Detailsof these processing steps are described below.

The first process step involves the synthesis of osmium intermediate,cis-bis(2,2′-bipyridyl)dichloroosmium(II), from commercially-availableosmium salt using the following scheme. The intermediate product isisolated through recrystallization in an ice bath.

The second process step involves the reaction between the osmiumintermediate and histamine to produce the desired osmium mediators. Thedesired product is then precipitated out from solution by addition ofammonium tetrafluoroborate.

These osmium mediators can also be easily converted to the oxidizedform, i.e., Os (III) using nitrosonium tetrafluoroborate. However, thisis unnecessary since the osmium reverts back to the reduced form atalkaline conditions during conjugation reactions and the affinity assaysdo not require the oxidized form of Os (III) for the detection on thebiosensor.

The free amino group of histamine in the osmium mediator (compound 5)was used to couple to the activated ester of drug derivative in generalto give drug-osmium conjugates. Similar osmium conjugates have beenprepared for HbAlc and HbA₀ peptides as described in U.S. Pat. No.6,262,264.

FIG. 18 illustrates an improved procedure for the synthesis of the keyintermediate, bis(2,2′-bipyridyl)-histamine-chloro-osmium mediator(compound 5). The original procedure involves the reaction of histaminewith cis-bis(2,2′-bipyridyl)dichloroosmium(II) in ethanol at a refluxcondition. However, poor yield of the resulting desired product led toan alternative synthetic route using a protected histamine derivative asthe starting material. Use of protecting groups in the organic chemistryis well-known in the art (“Protecting Groups in Organic Synthesis” by T.W. Green, John Wiley & Sons, 1981).

Thus, the primary amino group of histamine can be protected with asuitable protecting group, most preferably by using atert-butoxycarbonyl protecting group (t-BOC) or trifluoroacetamidogroup. The histamine dihydrochloride was reacted withdi-t-butyldicarbonate in THF to give di-t-BOC protected histaminederivative. The t-BOC group from the imidazole nitrogen was selectivelyremoved by reaction with triethyl amine in methanol. The mono protectedhistamine was coupled with Os^(II)(bPy)₂Cl₂ to give protected histaminecomplex (compound 4). The t-BOC group of protected histamine wasdeprotected by reaction with trifluoroactic acid to give osmium(bPy)₂(histamine)Cl (compound 5).

Several osmium histamine drug conjugates have been prepared. Arepresentative example is the reaction of osmium (bPy)₂(histamine)Cl(compound 5) with an amphetamine NHS ester (compound 8) as shown in FIG.19. The trifluoroacetamido group of the resulting osmium-amphetaminecomplex has been deprotected by reaction with 50 mM potassium carbonateto give amphetamine osmium conjugate (compound 10).

The osmium theophylline conjugate (compound 13) was prepared asillustrated in FIG. 20. Theophylline amine (compound 11) was preparedaccording to the procedure published in WO 87/07955. Theophylline amine(compound 11) was reacted with terephthalic acid di-N-hydroxysuccinimideester in the presence of triethyl amine to give theophyllineN-hydroxysuccinimide ester (compound 12). This activated ester wascoupled with osmium (bipy)₂(histamine)Cl (compound 5) in the presence oftriethyl amine to provide the osmium theophylline conjugate (compound13).

The PCP NHS ester (compound 14) was used to synthesize PCP-Osmiumcomplex (compound 15) which is shown in FIG. 21. The PCP NHS ester(Compound 14) was prepared according to the procedures published in U.S.Pat. No. 5,939,332.

The synthesis of osmium THC-2 conjugate (compound 17) is described inFIG. 22. The synthesis of THC-2 derivative (compound 16) is described inEP 0736529A1.

The synthesis of osmium THC-1 conjugate (compound 19), is described inFIG. 23. The synthesis of THC-1 derivative (compound 18) is described inJ. Org. Chem. 1986, 51, 5463-5465.

The synthesis scheme of osmium methotrexate conjugate is shown in FIG.24.

In one embodiment, the invention uses multi-osmium mediators. It wasdiscovered that the use of multi-osmium mediators improves the detectionsensitivity in the assays. Syntheses of osmium drug conjugates withmultiple osmium redox centers were designed. Multi-functionalizedaliphatic and aromatic linkers were designed to couple to osmiummediator amine with an additional different protected functionality.These mono-protected multi-osmium labels were deprotected and can beused to couple to drugs or other analytes of interest. As an example, adi-osmium THC-1 conjugate was prepared as shown in FIGS. 25-28. Thus,3,5-dihydroxy benzyl alcohol was reacted with suitably protected haloalkylating reagent, most preferably t-butyl bromo acetate in thepresence of a base, most preferably potassium carbonate to givedisubstituted product of compound 22. The benzyl alcohol functionalityof compound 22 was converted to a mesylate group followed by conversionto an azido group through a series of substitution reactions. The azidegroup of compound 24 was converted to an amino functionality byhydrogenation followed by protection as a trifluoroactamido groupproviding compound 26. The t-butyl ester functionalities were removed bytreatment with trifluoroacetic acid to provide the corresponding diacidcompound 27. This was converted to diacid chloride and reacted withosmium histamine amine of compound 5 to give the desired di-osmiumaromatic linked mono-trifluoroacetamide (compound 29). A monosubstitutedproduct was also isolated (compound 30) and evaluated for comparisonpurposes in the detection sensitivity measurement for electrochemicalassay. The trifluoroacetamido group of compound 29 can be removed underbasic conditions, most preferably aqueous potassium carbonate to givediosmium mediator 31 which can be coupled to an antigen (i.e. drugderivative; see FIG. 28) with the proper activating group.

Similarly a di-osmium complex with an aliphatic linker was alsoprepared. The aliphatic linker can be coupling to a suitably activatedantigen (e.g. drug derivative) as shown in FIG. 30.

In another embodiment of the invention an osmium complex with ahydrophilic linker was prepared as shown in FIGS. 30-32. The osmiumhistamine mediator with hydrophilic linker has been suggested toovercome assay development difficulties seen with the hydrophobicanalytes such as THC. A commercially available hydrophilic linker withproper functionalities is available (compound 33). This PEG linker has aprotected amino functionality and a free carboxylic acid. The acid groupof the PEG linker can be converted to an activated ester, preferably aN-hydroxysuccinimide ester and can be coupled to osmium histamine aminoderivative (compound 5) in the presence of a base, preferablytriethylamine to give compound 35. The t-BOC functionality of compound35 was removed under acidic conditions, preferably using trifluoroaceticacid. The free amino group of the osmium PEG linker (compound 36) can bereacted with an activated ester linked drug, as example, THC-2-NHS(compound 16) to give the osmium-PEG-THC conjugate (compound 37).

Osmium-PEG linker (compound 36) was coupled to methotrexate according toscheme shown in FIG. 33.

FIGS. 34-36 illustrate a synthetic scheme to prepare a tetra osmiumlabel. The intermediate bromo derivative, compound 23 can be reactedwith 3,5-dihydroxybenzyl alcohol in the presence of a base to give thecorresponding di-substituted product (compound 50). The benzyl alcoholgroup of compound 50 can be converted to the corresponding amine,compound 52, according to the similar procedure described above. Theamine group can be protected using a suitable protecting group, mostpreferably a trifluoroacetamido group and t-butyl ester groups, whichcan later be removed under acidic conditions. The resulting tetracarboxylic acid functionalities of compound 53 can be converted to thecorresponding acid chloride and can be coupled to the osmium histamineamine of compound 5 to give tetra-osmium aromatic label withtrifluoroacetamido protected amine. The amino group can be releasedunder basic conditions and the resulting compound 56 can be reacted withsuitably activated antigen (e.g. drug derivatives) to give theantigen-tetra-osmium conjugate.

In another embodiment of the invention, the synthesis of an osmiumdi-biimidazole histamine compound is described. This compound wasdeveloped to have a low redox potential to avoid potential interferentsfrom undesirable oxidizable species that may also be in the sample suchas ascorbic acid. The synthesis of di-biimidazole is shown in FIG. 37.

Thus glyoxal is reacted with ammonia to give biimidazole (compound 45).Dimethyl derivative of compound 45 can be reacted with methyl p-toluenesulfonate in the presence of a base, preferably sodium hydride to givecompound 46. This can be reacted with osmium trichloride in DMF at 180°C. to give osmium di-biimidazole dichloride (compound E). The couplingof compound 47 and histamine t-BOC (compound 4) in the presence of abase, preferably triethylamine provides compound 48. The t-BOC group ofcompound 48 can be removed under acidic conditions, preferably usingtrifluoroacetic acid to give compound 49. The free amino group of osmiumdi-biimidazole histamine (compound 49) can be coupled to activated esterof drug derivative to provide drug-osmium di-biimidazole histamineconjugates.

The osmium di-biimidazole histamine (compound 49) exhibited a low redoxpotential E_(1/2)˜540 mV vs Ag/AgCl. Mediators with low potentials arealso necessary for multi analyte measurements in as described in U.S.Pat. No. 6,294,062. In this case multiple electrochemical labels ormediators are needed each with different redox potentials spaced toallow each electrochemical label to be independently addressed.

Example 1 Electrochemical Assays of an Osmium-Theophylline Conjugate

An Os-theophylline conjugate was prepared as illustrated in FIG. 20.This redox reversible conjugate was evaluated with a series ofelectrochemical measurements designed to develop an assay response fortheophylline. IDA microelectrodes were fabricated as described hereinusing photolithographic techniques. The IDAs included gold electrodestructures with 50 finger pairs each having a width (W) of about 21 μmand a gap (W_(g)) of 10, 15, and 21 μm and length (b) of 6 mm. Each IDAalso contained two additional gold electrode regions for use as acounter and reference electrode. The electrochemical measurements wereperformed using a CH Instruments bipotentiostat model 802A or 832A.Bipotentiostatic amperometric measurements were made for IDAamplification and a single potential amperometric technique was used forenzyme amplification. Each measurement required about 20 μL samplepipette onto the electrode when using an external reference electrode or5-10 μl with an internal reference electrode of Ag/AgCl ink and acapillary roof over the active electrode structures.

FIG. 38 shows a CV of the osmium-theophylline conjugate on a 10 μm gap(W_(g)), 21μm (W) IDA electrode with 50 finger pairs. The CV showssymmetrical oxidation and reduction peaks and an E_(1/2) of about 125 mVvs. Ag/AgCl. From this the proper anodic and cathodic potentials can beselected for amperometric measurements and controlled with abipotentiostat.

FIG. 39 illustrates the oxidative steady state response to theosmium-theophylline conjugate measured at different concentrations on a21 μm gap (W_(g)), 21 μm (W) IDA electrode with 50 finger pairs. Abipotentiostat was used at the proper anodic and cathodic potentialsapplied to WE1 and WE2. This graph illustrates that even this largerdimensioned IDA reach steady stated in a few seconds.

FIG. 40 illustrates a dose response curve of the osmium-theophyllineconjugate at various concentrations on a 21 μm gap (W_(g)), 21 μm (W)IDA electrode with 50 finger pairs. The electrode potentials werecontrolled with a bipotentiostat. Again, it can be observed that theoxidation and reduction are approximately equal in absolute magnitude,which is indicative that no other species in the sample is interferingwith the current measurement at the electrodes.

It will be observed that the dose response curves herein display amultimeter bias. The term “multimeter bias” refers to a bias in the datacollected that is solely a result of the electrodes and instruments alsobeing connected to a high impedance multimeter, in this case a Fluke 87multimeter with an input impedance of 10 MOhm. For amperomentericmeasurements including bipotentiostatically controlled amperometricmeasurements, the bias is a constant I=V/R where V is the appliedpotential in volts for each respective electrode and R=10 MOhm. For theapplied potential of 0 mV vs. Ag/AgCl the bias would be 0 nA but for theapplied potential of 200 mV the bias would be 20 nA.

FIG. 41 is a plot illustrating the inhibition of the conjugate responsewith increasing concentrations of the antibody in solution. In theexample, the osmium-theophylline conjugate concentration was maintainedat about 25 μM, while the concentration of the antibody in solutionincreased. From the inhibition curve, it was determined that for thisexample, the optimal conjugate to antibody ratio is 2:1 since the slopeof the inhibition curve decreases significantly when antibodyconcentration increases further. This corresponds to the stoichiometricratio of 1:1 since antibodies are bivalent. It should also be noted thatthe response also shows a multimeter bias.

FIG. 42 is a theophylline assay run in serum calibrators and plots theIDA amplified current obtained for the osmium-theophylline conjugate.The test was run with a osmium-theophylline conjugate concentration ofabout 25 μM and antibody concentration of about 12.5 μM while varyingthe concentration of theophylline. The assay has a broad assay rangethat spreads from below the therapeutic range to the toxic region.

Example 2 Electrochemical Assays of an Osmium-Amphetamine Conjugate

An Os-amphetamine conjugate (10) was prepared as illustrated in FIG. 19.This redox reversible conjugate was evaluated in a series ofelectrochemical assays. Interdigitated array (IDA) microelectrodes werefabricated as described herein using photolithographic techniques. EachIDA contained 50 pairs of “fingers” each finger had a width of 21 μm anda gap width between the fingers of 15 μm. The electrochemicalmeasurements were performed using a CH Instruments bipotentiostat model802A or 832A. Bipotentiostatic amperometric measurements were made forIDA amplification and a single potential amperometric technique was usedfor enzyme amplification. Each measurement required about 20 μL samplepipette onto the electrode when using an external reference electrode or5-10 μl with an internal reference electrode of Ag/AgCl ink and acapillary roof over the active electrode structures

FIG. 43 shows a cyclic voltammogram of 100 μM Os-amphetamine conjugateprepared in a PBST solution. The figure shows a CV with a single fingerset and when both finger set are shorted together and used as theworking electrode. The CV shows symmetrical oxidation and reductionpeaks and an E_(1/2) of about 125 mV vs. Ag/AgCl. From this the properanodic and cathodic potentials can be selected.

FIG. 44 is a recycling CV of 100 μM osmium-amphetamine conjugate on a 15μm IDA electrode using a bipotentiostat to control the potentials. WE1was scanned from −100 to 400 mV while WE2 was held constant at −100 mV.The CV shows the mediator undergoing redox recycling. The CV shows asteady-state response of about 600 nA. or 6 nA/μM. The recycling CVshows that the oxidation and reduction current achieve have equal andopposite magnitudes. The current is amplified due to recycling. Tomeasure the recycling CV the WE2 was fixed at a reduction potential of−100 mV and WE1 was scanned between a reduction potential of −100 mV andan oxidation the potential of 400 mV. Recycling occurs when one fingerset is poised for oxidation and the other for reduction. Generally, aslong as the sweep rate is not too fast, the magnitude of the current isnot proportional to sweep rate. This differs from a normal CV where theresponse increased with sweep rate.

FIG. 45 is an osmium-amphetamine conjugate dose response on a IDA with a15 μm gap and 21 μm width. The osmium-amphetamine conjugate was preparedin PBST at concentrations from 0 μM to 100 μM. A CH Instrumentsbipotentiostat was used to make the measurements. The current observedat both working electrode #1 and working electrode #2 was approximatelyequal in absolute magnitude and slope (assuming subtraction of themultimeter bias). The plot was generated using a 15 μm IDA with aninternal Ag/AgCl reference electrode and WE1=300 mV and WE2=0 mV

The inhibition of the osmium-amphetamine conjugate was also evaluatedusing a bipotentiostat on an IDA with a 15 μm gap and 21 μm width. Theelectrodes were poised where WE1=250 mV and WE2=−150 mV vs. an internalreference electrode. The Os-amphetamine conjugate was mixed with varyingconcentrations of a monoclonal amphetamine antibody from Roche[<AMPH>M-2.17.22>] and a inhibition curve the optimal ratio of ratio ofconjugate to antibody was determined to be the stoichiometric ratio of2:1. This corresponds to the stoichometric ratio since the antibody isbivalent. This ratio was then used to demonstrate an amphetamine assayutilizing IDA amplification

FIG. 46 shows a plot of an amphetamine assay. Varying concentrations ofd,l-amphetamine drug were mixed with a fixed concentration of antibodyfollowed by a fixed concentration of conjugate. The final solutionmatrix contained 20 μM osmium amphetamine conjugate, 10 μM antibody, andamphetamine concentrations between 0 and 25 μM. Each solution was mixedand immediately transferred onto an IDA electrode to measure the currentresponse using a bipotentiostat. The measured oxidative and reductivecurrent responses are plotted against the drug concentration to producethe assay dose response curve. The current response from the conjugateincreased as more drug was added and bound antibody resulting in lessconjugate inhibition. The assay dose response curve covers the rangerequired for amphetamine drugs of abuse assay which has a SAMHSAmandated cutoff concentration of 1000 ng/mL or 6.7 μM. The amphetamineresponse was evaluated using a bipotentiostat on an IDA with a 15 μm gapand 21 μm width. The electrodes were poised where WE1=250 mV andWE2=−150 mV vs. an internal reference electrode.

Example 3 Osmium-Biotin Model System with 2 μm IDA

Interdigitated array electrodes were custom fabricated for RocheDiagnostics by Premitec, Inc., Raleigh, N.C. Each IDA contained 750pairs of electrodes each 6 mm in length with a gap and width of 2 μm.The interdigitated region for these electrodes totaled 36 mm². Thislarge dimension was selected based on practical considerations ofachieving lower detection limits Instruments, especially a portablehandheld bipotentiostat, have limitations as to the minimum currentsthat can be detected. At the time, an assumption was made that 1 nAwould be the lowest current that would be measured for an assay due tonoise considerations of a handheld device. Using this information alongwith predicted current responses, the dimensions of the electrodes weredetermined to allow a sizeable improvement in immunoassay sensitivity.In addition these electrodes were also fabricated to compare thecalculated sensitivity to that actually observed to demonstrate thatimproved amplification as would be predicted by Equation 1.

An osmium-biotin conjugate (compound 57 shown below) was preparedaccording to procedures similar to as describe in U.S. Pat. No.6,262,264

A microcentrifuge tube was charged with 20 μl of biotin in the followingconcentrations: 0, 1.25, 2.5, 3.75, 5, 7.5, and 10 μM. To each of thesesolutions were added 20 μL of 1.25 μM streptavidin and 40 μL PBST. Toeach solution, 20 μL of 5 μM streptavidin was added, mixed briefly (˜2s), and pipetted between the capillary space built on a 2 μM IDAelectrode. A bipotentiostatic measurement was immediately initiatedcontrolling WE1 at 250 mV and WE2 at 0 mV vs. Ag/AgCl.

FIG. 47 shows the recycling cyclic voltamogram (CV) steady-stateresponse obtained with 25 μM of osmium free mediator(bis(2,2′-bipyridyl)imidazole chloro osmium (III)dichloride) on a planarIDA electrode with 750 finger pairs with W and Wg of 2 μm. The recyclingCV was run with WE1 being the generator scanned from −100 mV to 400 mVand WE2 the collector held at −100 mV. This response shows excellentamplification and efficiency with the steady-state response of about3800 nA or 152 nA/μM.

FIG. 48 shows a dose response curve of the osmium biotin conjugate 0-5μM on a planar IDA electrode with 750 finger pairs with W and W_(g) of 2μm. The measurements controlled WE1=250 mV and WE2=0 mV with a CHIInstruments bipotentiostat. The slope of the oxidation and reductivemeasurements are approximately equal and opposite indicating good redoxrecycling efficiency. The slope of about 126 nA/μM with the osmiumbiotin is a very good response only slightly less then the 152 nA/μMcalculated for osmium free mediator from the CV of FIG. 47.

Data was collected at 0.5 sec intervals for 40 seconds. The assayresponse was evaluated at several time points and giving similar resultsat all times including the 0.5 second time point. FIG. 49 shows thebiotin assay response at time=0.5, 2, and 10 seconds. The assay reagentswere selected for an assay range of 0-1 μM which was achieved. A typicalplot showing a steady-state current vs. time is shown in FIG. 50. Thedata shows that a steady-state response is achieved almostinstantaneously from the onset of the applied potentials. Steady-stateis achieved when the oxidized and reduced species generated onrespective electrodes are equal.

These results were collected using a 2 μM IDA electrode that wasprepared at Premitec Inc, Raleigh, N.C. and was from wafer ID#0702HIDA1. . . 14. The final assay concentrations were 1 μM of the osmium biotinconjugate, 0.25 μM streptavidin and biotin from 0-2 μM. This assay wasperformed using a CHI-802A bipotentiostat from CH Instruments, Austin,Tex.

FIG. 51 and FIG. 54 show normal CVs that were performed with both themono-osmium-aromatic trifluoroacetomido protected linker (compound 30)and the di-osmium-aromatic linker (compound 31) respectively. The CVsshow that the synthesized mediators were redox reversible compounds witha reasonably low E_(1/2) potential of about 20-40 mV vs Ag/AgCl. Bothwere run at a concentration of 200 μM on planar IDA electrodes with 50finger pairs with W=21 μm and W_(g)=15 μm.

FIG. 53 show a comparison of dose responses for the mono-osmium-aromatictrifluoroacetomido protected linkers (compounds 30 and 31), and osmiumfree mediator (bis(2,2′-bipyridyl)imidazole chloro osmium(III)dichloride) all run on planar IDA electrodes with 50 finger pairswith W=21 μm and Wg=15 μm. The osmium free mediator and themono-osmium-aromatic trifluoroacetomido protected linker gave similarresponse and the di-osmium-aromatic linker gave a slightly improvedresponse. The response of the di-osmium compound was not 2× that onemight initially expect with twice the number of redox sites. Somejustification for a lower response may be that the larger size (MW) ofthe di-osmium complex slows the diffusion between electrodes and/or thaton average only one site of the di-osmium complex is actually oxidizedand reduced (only one osmium center is oxidized or reduced at any time).In any case based upon the CV and a slightly improved dose responsecurve, this new conjugate proved to be a viable alternative to themediators with single osmium centers.

FIG. 54 show the CV of di-osmium-THC1 conjugate (compound 32) at aconcentration of 200 μM run on planar IDA electrodes with 50 fingerpairs with W=21 μm and W_(g)=15 μm. The conjugate stock was prepared inPBST from 0.75 mg of the compound without the use of an organic solvent.The CV shows that the mediator is reversible but the reductive peakappears to be slightly smaller than the oxidation peak's currents. Theresponse is about 50% lower than the di-osmium-aromatic linker (compound31). The decreased response seems to be indicative of the hydrophobicnature of the THC molecule.

FIG. 55 is an osmium-PEG-THC-2 (compound 37) dose response curve onplanar IDA electrodes with 50 finger pairs with W=21 μm and W_(g)=15 μm.The measurements were controlled with a CHI Instruments bipotentiostatWE1=250 mV and WE2=0 mV. The conjugate stock solution was dissolved inPBST without the use of organic solvents. Serial dilutions were madebetween 6.25 μM to 500 μM in PBST. 10 μl was and applied into thecapillary built over the electrode cell. Similar to the di-osmium-THC1conjugate (compound 32) the reductive response (slope) is slightlysmaller than the oxidation slope. Both slopes are about 10 times smallerthat seen with the osmium free mediator. The assay data shown is theresult 5 seconds after applying the potentials. The results are theaverage of 4 replicates.

FIG. 56 is an osmium-PEG-THC-2 (compound 37) dose response curve withenzyme amplification. The slope of the enzyme amplified conjugateresponse is improved over the IDA amplification. Also noted is that withthe addition of 5% hydroxypropyl-β-cyclodextrin to overcome possibleconcerns with the hydrophobic nature of the THC, a significant increaseis seen in the response.

FIG. 57 shows the CV of osmium-PEG-methotrexate (compound 38) at aconcentration of 150 μM run on planar IDA electrodes with 50 fingerpairs with W=21 μm and W_(g)=15 μm. The conjugate stock was prepared inPBST at 150 μM from 0.49 mg of the compound without the use of anorganic solvent. The CV shows a symmetrical reversible redox peaks. Incomparison CVs of other mediators, one must take into account the largerfinger width of 21 μM and the lower concentration of conjugate use forthis CV.

FIG. 58 is an osmium-PEG-methotrexate (compound 38) dose response curveon planar IDA electrodes with 50 finger pairs with W=21 μm and W_(g)=21μm. The measurements were controlled with a CHI Instrumentsbipotentiostat WE1=0.25 mV and WE2=0 mV. Dilutions were made in PBSTfrom the 150 μM stock to prepare (25, 10, 5, 2.5 and 1.25 μMconcentrations). 20 μL of the solutions were applied to the electrodeinto the capillary built over the electrode cell using an externalAg/AgCl reference electrode. The 20 μL volume was needed to bridge thegap between the sample in the capillary and the external capillarylocated just outside the capillary region. The slope of conjugateresponse for this mediator was larger than that of the osmium-PEG-THC2response. The results are the average of 4 replicates.

Additional examples are described in an article entitled “The LatestDevelopment in Biosensor Immunoassay Technology for Drug Assays” whichis incorporated by reference in its entirety.

Synthesis of Electrochemical Mediator Labels

All solvents were from J. T. Baker. Analytical reverse phase HPLCanalyses were performed on an Agilent HP1100 LC/MS system configuredwith a diode-array detector and a quaternary pump. The LC/MS analyseswere performed with a Vydac 218TP54 column (300A, 5μ; C18, 4.6 mm×250mm) equipped with a Phenomenex KJO-4282 guard kit with AJO-4287(C-180DS) cartridge. Chromatographic stream ported post-column into theMS detector. The MSD utilized was run in electrospray positive mode “ES(+) mode”.

HPLC fractions were lyophilized. Acetonitrile was evaporated underreduced pressure followed by freezing of the aqueous residue using, forexample, a dry ice/acetone bath, followed by freeze-drying using alyophilizer. The residue was purified by preparative RP-HPLC to give10.2 mg (6.6×10⁻³ mmol, 20%) of THC-osmium PEG derivative (37), LC/MSM+H 1501.6.

Preparative reverse phase HPLC used a Varian Dynamax radial compressioncolumn with 1) R00083221C (Microsorb 60-8, C-18, 250 mm×21.4 mm) with aVarian Dynamax (Rainin) guard module R00083221G (C-18, 8μ) or 2)R00083241C (Microsorb 60-8, C-18, 250 mm×41.4 mm) with a Varian Dynamax(Rainin) guard module R00083241G (C-18, 8μ). The HPLC work was performedusing a gradient system of water-acetonitrile containing 0.1%trifluoroacetic acid.

Amphetamine NHS ester (compound was prepared as described in “DualAnalyte Immunoassay”, EP 0574782A2. The THC-1 NHS ester (compound 18) isa short linked derivative prepared as described in “Reagents for theDetermination of Drugs”, EP 0386644. The THC-2 NHS ester (compound 16)is a long chain derivative prepared as described in “Novel CannabinolDerivatives and Improved Immunoassay”, EP 0276732A2. Theophylline amine(compound 11) was prepared according to the procedure published in PCTWO 87/07955. Theophylline NHS ester (compound 12) was prepared byreaction of theophylline amine (compound 11) with terephthalic aciddi-N-hydroxysuccinimide ester in the presence of triethylamine. TheO—(N-Boc-2-aminoethyl)-O—(N-diglycolyl)-2-aminoethyl)hexaethyleneglycol(compound 33) was purchased from Nova Biochem through VWR.

Preparation of bis(2,2′-bipyridyl)dichloro osmium

To a mixture of 4.18 g (8.6 mmol) of potassium hexachloroosmiate and3.05 g (19.5 mmol) 2,2′-dipyridyl was added 100 mL of dimethylformamide.The reaction mixture was heated to reflux for 1 h and cooled to roomtemperature. The resulting reaction mixture was filtered and the residuewas washed with 5 mL of DMF. The filtrate was allowed to stir at roomtemperature and a solution of 4.76 g (0.027 mol) sodium dithionate in430 mL of water was added to the reaction mixture dropwise. Theresulting reaction mixture was placed in an ice-bath to precipitate thedesired product. This resulting solid was collected and washed two timeswith 15 mL of water followed by two times with 15 mL of ether. Theresulting brown solid was dried at 50° C. under vacuum (0.5 mm Hg) togive 4.3 g (7.4 mmol, 87%) of desired product as a dark brown solid.

Preparation of4-(2-tert-Butoxycarbonylamino-ethyl)-imidazole-1-carboxylic acidtert-butyl ester

A mixture of 3.68 g (20 mmol) of histamine dihydrochloride, 160 mLacetonitrile, 14 mL (0.10 mol) of triethylamine, and 13.1 g (0.060 mol)of di-tert-butyldicarbonate was allowed to stir at room temperature forthree days. The reaction mixture was concentrated, and the residue waswashed with 150 mL of hexane. The residue was washed three times with100 mL of ether. All the ether parts were combined and concentrated togive as a white powder (LC/MS M+NA 334.1).

Preparation of [2-(1H-Imidazol-4-yl)-ethyl]-carbamic acid tert-butylester

To 4-(2-tert-Butoxycarbonylamino-ethyl)-imidazole-1-carboxylic acidtert-butyl ester, were added 100 mL of methanol and 800 mL (5.73 mol) oftriethylamine The reaction mixture was allowed to stir at roomtemperature for four days and concentrated to oil. To the residue wereadded 40 mL of ether and 80 mL of hexane. This mixture was allowed tostand at room temperature resulting in precipitation of product, as awhite solid, which were collected. The yield was 1.7 g (8.0 mmol, 40%)(LC/MS M+H 212.1).

Preparation of Osmium dibipyridyl t-Boc Histamine (Compound 4)

To a mixture of 300 mg (0.52 mmol) of bis(2,2′-bipyridyl)dichloro osmiumand 268 mg (1.26 mmol) of histamine t-Boc was added 54 mL of ethanolfollowed by 1.6 mL (11.4 mmol) of triethylamine. The mixture was allowedto stir at 80° C. for five days and concentrated. The residue waspurified by preparative HPLC using a gradient run with water andacetonitrile containing 0.1% trifluoroacetic acid to give 250 mg (0.32mmol, 61%) of osmium dibipyridyl t-Boc Histamine (compound 4) as a brownpowder (LC/MS M+H 749.1).

Preparation of Bis(2,2′-bipyridyl)-histamine-chloro-osmium[Osmium(bP_(y))₂(histamine)Cl] (Compound 5)

To 50 mg (0.063 mmol) of osmium dibipyridyl t-Boc Histamine (compound 4)were added 2 mL methylene chloride and 2 mL of trifluoroacetic acid. Thereaction mixture was allowed to stir at room temperature for 20 minutesand concentrated under reduced pressure. To the residue 5 mL ofmethylene chloride was added and concentrated. This procedure ofaddition of 5 mL methylene chloride followed by concentration wasrepeated four more times and the total residue was dried to give 40 mg(0.058 mmol, 93%) of (compound 5) as a brown powder, (LC/MS M+H 649).

Preparation of Amphetamine-Osmium Histamine TFA Protected Conjugate(Compound 9)

To 82 mg (0.104 mmol) of osmium dibipyridyl t-Boc histamine (compound 4)were added 1.5 mL of trifluoroacetic acid and 0.5 mL of1,2-dichloroethane. The resulting reaction mixture was allowed to stirat room temperature for 30 minutes and then concentrated. To theresulting residue was added 5 mL of methylene chloride, which was thenconcentrated. The above process of addition of 5 mL of 1,2-methylenechloride and concentration was repeated three additional times and thetotal dried under reduced pressure for 2 h. To the residue were added 1mL anhydrous DMF and 0.2 mL (1.43 mmol) of triethylamine. The reactionmixture was allowed to stir while a solution of 50 mg (0.09 mmol) ofamphetamine NHS ester in 1 mL of 1, 2 dichloroethane and 0.5 mL of DMFwere added dropwise. The resulting reaction mixture was allowed to stirat room temperature 18 hours and then concentrated under reducedpressure. The residue was purified by preparative reverse phase HPLC togive 68 mg (0.060 mmol, 67%) of amphetamine-osmium trifluoroacetamideprotected compound (compound 9) as dark brown solid, LC/MS M+H 1083.63.

Preparation of Osmium Amphetamine Conjugate (Compound 10)

To 60 mg (0.053 mmol) of amphetamine-osmium histamine TFA protectedconjugate (compound 9) were added 25 mL of 50 mM potassium carbonate and10 mL of methanol. The reaction mixture was allowed to stir at roomtemperature for three days and then concentrated. The residue waspurified by preparative reverse phase HPLC to give 13.6 mg (0.013 mmol,25%) of the osmium conjugate (compound 10), LC/MS M+H 987.2. Thestarting material amphetamine-osmium histamine TFA protected conjugate(compound 9) (18.3 mg) was also recovered.

Preparation of Osmium THC-2 histamine Conjugate (Compound 17)

To 54 mg (0.068 mmol) of osmium dibipyridyl-t-Boc histamine (compound 4)were added 1.5 mL trifluoroacetic acid and 0.5 mL of methylene chloride.The reaction mixture was allowed to stir for 30 minutes andconcentrated. Methylene chloride, 5 mL, was added and resulting solutionconcentrated. The above procedure of methylene chloride addition andconcentration was repeated three more times. To the resulting residue, 1mL of anhydrous DMF was added followed by 200 μL (1.43 mmol) oftriethylamine. The reaction mixture was allowed to stir at roomtemperature and a solution of 30 mg (0.060 mmol) of THC-2 NHS derivative(compound 16) in 0.5 mL of anhydrous DMF and 1 mL of methylene chloride.The reaction mixture was allowed to stir at room temperature 18 hoursand concentrated. The residue was purified by preparative reverse phaseHPLC to give 33.3 mg (0.031 mmol) of osmium THC-2 histamine conjugate(compound 17) as brown powder, LC/MS M+H 1034.2.

Osmium methotrexate conjugate (Compound 21)

To 57 mg (0.125 mmol) of methotrexate (Sigma) was added 1 mL ofanhydrous DMF followed by 22 mg (0.15 mmol) of 4-nitrophenol and 27 mg(0.13 mmol) of N,N′-dicyclohexylcarbodiimide. The resulting reactionmixture was allowed to stir at room temperature for 4 hours and theresulting methotrexate activated ester (compound 20) was used in situ inthe next step without isolation.

To 100 mg (0.14 mmol) of osmium dibipyridyl t-Boc histamine (compound 4)was added 1 mL of trifluoroacetic acid. The resulting reaction mixturewas allowed to stir at room temperature 30 minutes and concentratedunder reduced pressure. To the residue 5 mL of methylene chloride wasadded and concentrated. The addition of 5 mL methylene chloride andconcentration process was repeated four more times. To the residue 1 mLof DMF was added followed by 200 μL (1.43 mmol) of triethylamine. Thereaction mixture was allowed to stir at room temperature under argonatmosphere and the activated ester of methotrexate prepared as above(compound 20) was added dropwise. The reaction was allowed to stir for18 hours at room temperature under argon atmosphere and concentratedunder reduced pressure. The residue was purified by preparative reversephase HPLC to give 52.5 mg (0.046 mmol, 32%) of the osmium methotrexateconjugate (compound 21) as a brown powder, LC/MS M+H 1086.2.

Preparation of Theophylline Osmium histamine conjugate (Compound 13)

This was prepared by similar method starting with theophylline NHS ester(compound 12) as described for example in the preparation of osmiumTHC-2 histamine conjugate (compound 17).

Preparation of PCP-Osmium histamine conjugate (Compound 15)

This was prepared by similar method starting from PCP NHS ester(compound 14) as described for example in the preparation of osmiumTHC-2 histamine conjugate (compound 17).

Preparation of THC-1 Osmium histamine conjugate (Compound 19)

This was prepared by similar method starting from THC-1 NHS ester(compound 18) as described for example in the preparation of osmiumTHC-2 histamine conjugate (compound 17). Solubility of this mediator inPBST was poor and required the use of DMF.

Preparation of(3-tert-Butoxycarbonylmethoxy-5-hydroxymethyl-phenoxy)-acetic acidtert-butyl ester (Compound 22)

To 5 g (35 mmol) of 3,5-dihydroxybenzyl alcohol were added 250 mL of dryDMF, 11.85 mL (80 mmol) of t-butylbromoacetate, 14.8 g (107 mmol) ofanhydrous potassium carbonate, and 34.5 g (105 mmol) of cesium carbonatefollowed by 3 g of 4 Å molecular sieves. The resulting reaction mixturewas allowed to stir at 80° C. under argon atmosphere. The reactionmixture was allowed to cool to room temperature, filtered, and theresidue was washed with 200 mL of ethyl acetate. All the filtrate werecombined and concentrated to dryness. The residue was redissolved in 150mL of diethyl ether, washed three times with 200 mL of water andconcentrated. The residue was purified by silica gel columnchromatography using 70% diethyl ether in hexane to give 7.5 g (20 mmol,57%) of (compound 22) as a colorless gum (LC/MS M+Na 391.1).

Preparation of(3-Bromomethyl-5-tert-butoxycarbonylmethoxy-phenoxy)-acetic acidtert-butyl ester (Compound 23)

To 1 g (2.7 mmol) of (compound 22) was added 48 mL of methylene chlorideand cooled to −40° C. followed by 640 μL (4.58 mmol) of triethylamine,400 μL (5.16 mmol) of methanesulphonyl chloride. The resulting reactionmixture was allowed to stir at −40° C. for 3 h. To the reaction mixturewere added 32 mL of freshly distilled THF and 800 mg (9.21 mmol) oflithium bromide. The reaction mixture was allowed to stir at 4° C. for18 hours, and then concentrated to dryness. This was dissolved in 50 mLof methylene chloride and 20 mL of water. The organic layer wasseparated and the aqueous layer was extracted with four times with 30 mLof methylene chloride. The combined organic layers were dried (Na₂SO₄)and concentrated to give 1.16 g (2.68 mmol, 99%) of (compound 23).

Preparation of(3-Azidomethyl)-5-tert-butoxycarbonylmethoxy-phenoxy)-acetic acidtert-butyl ester (Compound 24)

To 1.16 g (2.68 mmol) of the bromo derivative (compound 23) were added30 mL of anhydrous DMF and 1.79 g (27.5 mmol) of sodium azide at 50° C.under an argon atmosphere for 72 hours. The reaction mixture was cooledto room temperature, filtered, and the residue was concentrated todryness. To the residue 50 mL of ethyl acetate and 25 mL of water wereadded. The organic layer was separated and the aqueous layer wasextracted with 25 mL of ethyl acetate. The organic layers were combined,dried (Na₂SO₄) and concentrated to give 1.04 g (2.64 mmol, 99%) of theazido compound (compound 24) as a gummy white solid, LC/MS M+Na 416.1.

Preparation of(3-Aminomethyl-5-tert-butoxycarbonylmethoxy-phenoxy)-acetic acidtert-butyl ester (Compound 25)

To 2.9 g (7.3 mmol) of (compound 24) were added 140 mL of ethanol, 4.5 g(71.3 mmol) of ammonium formate and 1.39 g of 10% Pd—C. The resultingreaction mixture was allowed to stir at room temperature for 18 hoursand filtered through CELITE®. The residue was washed with 50 mL ofethanol. The filtrate was concentrated and redissolved in 150 mL ofchloroform. This was washed three times with 75 mL of DI water, dried(Na₂SO₄) and concentrated to give 2.4 g (6.53 mmol, 89%) of the aminoderivative (compound 25) as a off-white semisolid, LC/MS; M+Na 390.1,2M+1 735.3.

Preparation of{3-tert-Butoxycarbonylmethoxy-5-[2,2,2-trifluoro-acetylamino)-methyl]-phenoxy}-aceticacid tert-butyl ester (Compound 26)

To 862 mg (2.34 mmol) of (compound 12) were added 20 mL of freshlydistilled THF, 1 mL (7.17 mmol) of triethylamine and 426 μL (3.57 mmol)of ethyltrifluoroacetate. The resulting reaction mixture was allowed tostir at room temperature for 18 hours. This was concentrated to drynessand dissolved in 50 mL of chloroform. The organic layer was washed threetimes with 50 mL of water, dried (Na₂SO₄) and concentrated. The residuewas purified by silica gel column chromatography using to give 604 mg(1.3 mmol, 56%) of trifluoroacetyl protected product (compound 26) as awhite gummy solid LC/MS M+NA 486.

Preparation of{3-Carboxymethoxy-5-[(2,2,2-trifluoro-acetylamino)-methyl]-phenoxy}-aceticacid [Aromatic trifluoroacetamido protected linker] (Compound 27)

To 500 mg (1.07 mmol) of (compound 26) were added 20 mL of methylenechloride and 20 mL of trifluoroacetic acid. The reaction mixture wasallowed to stir at room temperature for 72 hours and concentrated. Tothe residue was added 30 mL methylene chloride and concentrated. Theabove process of addition of 30 mL methylene chloride and concentrationwas repeated three more times to give 376 mg (1.07 mmol, 99%) of thediacid derivative (compound 27) as a white solid, LC/MS M+NA 374.

Preparation of{3-Chlorocarbonylmethoxy-5-[(2,2,2-trifluoro-acetylamino)-methyl]-phenoxy}-acetylchloride (Compound 28)

To 51.2 mg (0.145 mmol) of (compound) were added 3 mL of methylenechloride and 205 μL (2.33 mmol) of oxalyl chloride and 10 mL ofanhydrous DMF. The reaction mixture was allowed to stir at roomtemperature for 18 hours and concentrated. To the residue was added 5 mLof methylene chloride and concentrated.

The above process of addition of 5 mL methylene chloride andconcentration was repeated three more times to give the diacidchloride(compound 28). This was used in the next step without furtherpurification.

Preparation of Di-Osmium Dibipyridyl Histamine TrifluoroacetamidoProtected aromatic linker (Compound 29)

Osmium dibipyridyl t-Boc histamine (compound 4), 320 mg (0.407 mmol) wasdissolved in 8 mL of methylene chloride and 8 mL of trifluoroaceticacid. After stirring the resulting mixture at room temperature for 20minutes, the solvents were removed. Methylene chloride was then addedand then removed under vacuum. The addition and removal of methylenechloride was repeated three more times. Then the resulting solid wasdissolved in 3 mL of methylene chloride and was allowed to stir at roomtemperature. A solution of all of (compound 28) (prepared above) in 3 mLof methylene chloride was added to the reaction mixture followed by theaddition of 2 mL (14.3 mmol) of triethylamine. The reaction mixture wasallowed to stir at room temperature under argon atmosphere for threedays. Then the mixture was concentrated and purified by preparativereverse phase HPLC to give 55.9 mg (0.033 mmol, 23%) of the di-osmiumaromatic trifluoroacetamido protected complex (compound 29) (LC/MS M+H1614.3) and 71.8 mg (0.070 mmol, 48%) of the mono-osmium aromatictrifluoroacetamido protected complex. (compound 30) (LC/MS M+H 983.2)with 69.2 mg recovery of osmium(bPy)₂(histamine)Cl (compound 5).

Preparation of Di-Osmium dibipyridyl Histamine aromatic linker[Di-Osmium aromatic linker] (Compound 31)

To 53.6 mg (0.031 mmol) of di-osmium aromatic trifluoroacetamidoprotected complex (compound 29) were added 25 mL of 50 mM potassiumcarbonate and 10 mL of methanol. The reaction mixture was allowed tostir at room temperature for three days and concentrated. The residuewas purified by preparative reverse phase HPLC to give 24 mg (0.015mmol, 47%) of di-osmium dibipyridyl histamine aromatic linker (compound31) as a brown powder, LC/MS M+H 1519.2.

Preparation of Di-Osmium dibipyridyl Histamine THC-1 conjugate[Di-Osmium THC-1 Conjugate] (Compound 32)

To 11 mg (6.9×10⁻³ mmol) of di-osmium dibipyridyl histamine aromaticlinker (compound 31) were added 1.96 mL of DMF, 196 μL (1.40 mmol) oftriethylamine and 7.86 mg (0.0178 mmol) of THC-1-NHS ester derivative(compound 18). The mixture was allowed to stir at room temperature underargon atmosphere for 18 hours and concentrated. LC/MS indicated desiredproduct formation, (LC/MS M+H 1846.4). The above reaction was repeatedby mixing 8 mg (5.03×10⁻³ mmol) of di-osmium dibipyridyl histaminearomatic linker (compound 31), 1.43 mL DMF, 143 μL (1.01 mmol) oftriethylamine and 5.72 mg (0.012 mmol) of THC-1-NHS (compound 18). Thereaction mixture was allowed to stir at room temperature for 18 hoursand concentrated. Both of the reaction products were mixed and purifiedby preparative reverse phase HPLC to give 8.7 mg (4.5×10⁻³ mmol, 11%) ofdi-osmium THC-1 conjugate (compound 32) as a brown powder, (LC/MS, M+H1846.4).

Preparation of Osmium-PEG linker t-Boc protected (Compound 35)

To 120 mg (0.20 mmol) ofO—(N-Boc-2-aminoethyl)-O—(N-diglycolyl)-2-aminoethyl hexaethylene glycol(compound 33), (Nova Biochem) were added 2 mL of methylene chloride, 128mg (0.67 mmol) of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride, 72 mg (0.62 mmol) of N-hydroxysuccinimide. The reactionmixture was allowed to stir at room temperature for 18 hours. Theresulting activated PEG NHS ester (compound 34) was used in situ in thenext step without isolation.

To 120 mg (0.15 mmol) of Osmium dibipyridyl t-Boc Histamine (compound 4)was added 2.5 mL of trifluoroacetic acid and the resulting mixture wasallowed to stir at room temperature for 1 hour. The reaction mixture wasconcentrated and 15 mL of methylene chloride was added and concentratedto dryness. To the residue 1.5 mL of DMF was added followed by 500 μL(mmol) of triethylamine. The reaction mixture was allowed to stir atroom temperature and the solution of activated PEG NHS ester (compound34) was added dropwise to the reaction mixture. The reaction mixture wasallowed to stir at room temperature for 18 hours and concentrated. Theresidue was purified by preparative reverse phase HPLC to give 93 mg(0.074 mmol, 36%) of Osmium PEG linker t-Boc protected (compound 35) asa brown powder, LC/MS M+H 1216.4.

Preparation of Osmium PEG linker (Compound 36)

To 90 mg (0.071 mmol) of osmium PEG linker t-Boc protected (compound 35)was added 2 mL of trifluoroacetic acid. The resulting reaction mixturewas allowed to stir at room temperature for 40 minutes and concentratedto give 81 mg (0.070 mmol, 99%) of the osmium PEG linker (compound 36)as a dark brown thick gum, LC/MS M+H 1116.2.

Preparation of Osmium PEG THC-2 Conjugate (Compound 37)

To 39 mg (0.033 mmol) of osmium PEG linker (compound 36) were added 1 mLof DMF and 200 μL (1.43 mmol) of triethylamine. The reaction mixture wasallowed to stir at room temperature under argon atmosphere and asolution of 16 mg (0.032 mmol) of THC-2 NHS ester (compound 16) in 1 mLof methylene chloride was added dropwise to the reaction mixture. Thereaction was allowed to stir at room temperature for 18 hours andconcentrated under reduced pressure. The residue was purified bypreparative reverse phase HPLC to give 10.2 mg (6.6×10⁻³ mmol, 20%) ofosmium PEG THC-2 conjugate (compound 37), LC/MS M+H 1501.6.

Osmium PEG Methotrexate Conjugate (Compound 38)

To 19 mg (0.041 mmol) of methotrexate (Sigma) was added 0.4 mL ofanhydrous DMF followed by 8 mg (0.06 mmol) of 4-nitrophenol and 9 mg(0.043 mmol) of N,N′-dicyclohexylcarbodiimide. The resulting reactionmixture was allowed to stir at room temperature for 4 hours and theresulting activated ester (compound 20) was used in situ in the nextstep without isolation.

To 22.9 mg (0.019 mmol) of osmium PEG linker (compound 36) was added 500μL of DMF followed by 100 μL (0.71 mmol) of triethylamine. The reactionmixture was allowed to stir at room temperature and the solution ofmethotrexate activated ester prepared above (compound 20) was addeddropwise. The reaction mixture was allowed to stir at room temperaturefor 18 hours and concentrated. The residue was purified by preparativereverse phase HPLC to give 7.8 mg (4.9×10⁻³ mmol, 25%) of osmium PEGmethotrexate conjugate (compound 38), LC/MS M+H 1554.5.

Preparation of 4-amino-hepanedioic acid diethyl ester hydrochloride(Compound 39)

To 2 g (8.6 mmol) of diethyl 4-oxopimelate was added 20 mL of methanol,followed by 6.7 g (86 mmol) of ammonium acetate, 713 mg (8.6 mmol) ofsodium acetate, and 5 mL of glacial acetic acid. The reaction mixturewas allowed to stir at room temperature for 18 hours and concentrated.To the residue 150 mL ethyl acetate and 100 mL of aqueous saturatedsolution of sodium bicarbonate was added. The organic layer wasseparated and the aqueous layer was extracted with an additional 100 mLof ethyl acetate. Organic layers were combined and washed two times with100 mL of saturated sodium bicarbonate, dried (Na₂SO₄) and concentratedto give an oil. To the oil 5 mL of 2M HCl in diethylether was added.White solid precipitated out which was filtered to give 1.2 g (4.48mmol, 52%) of the amino product as a hydrochloride salt (compound 39).

Preparation of 4-tert Butoxycarbonylamino-heptanadioic acid diethylester (Compound 40)

To 500 mg (1.86 mmol) of the amino product (compound 39) was added 15 mLof methylene chloride followed by 1.2 mL (8.5 mmol) of triethylamine. Tothe reaction mixture 646 mg (2.95 mmol) of di-t-butyldicarbonate wasadded followed by 25 mg (1.12 mmol) of 4-dimethylaminopyridine. Thereaction mixture was allowed to stir at room temperature for 18 hoursand concentrated under reduced pressure. To the residue 150 mL ofchloroform was added and washed with two times with 100 mL of water. Theorganic layer was dried (Na₂SO₄) and concentrated to give an oil. Thiswas purified by silica gel column chromatography using 8:2 hexane:ethylacetate to give 396 mg (1.19 mmol, 64%) of the 4-tertButoxycarbonylamino-heptanadioic acid diethyl ester product (compound40).

Preparation of 4-tert-Butoxycarbonylamino-haptanedioic acid (Compound41)

4-tert Butoxycarbonylamino-heptanadioic acid diethyl ester (compound40), 380 mg (1.14 mmol), was dissolved in THF containing 3 mL methanol.To the reaction mixture a solution of 481 mg (11.5 mmol) of lithiumhydroxide hydrate in 6 mL of water was added and the reaction mixturewas allowed to stir at room temperature for 18 hours. This wasconcentrated under reduced pressure. Five mL of water was added and thepH of the solution was adjusted to pH-5 by using conc. H₃PO₄. Thereaction mixture was extracted with 3 times 75 mL of ethyl acetate. Thecombined organic layers were dried (Na₂SO₄) and concentrated to give 310mg (1.12 mmol) of (compound 41) as white powder [LR-MS-ER (−) (M−H274.2)].

Preparation of 4-tert-Butoxycarbonylamino-heptanedioic acidbis-(2,5-dioxo-pyrrolidin-1-yl)ester (Compound 42)

To 44 mg (0.15 mmol) of 4-tert-Butoxycarbonylamino-haptanedioic acid(compound 41) were added 5 mL of methylene chloride, 76 mg (0.39 mmol)of 1,3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride and 46 mg(0.39 mmol) of N-hydroxysuccinimide. The reaction mixture was allowed tostir at room temperature for 18 hours. 15 mL of methylene chloride wasadded. The organic layer was washed with two times with 15 mL of water,two times with 15 mL of saturated sodium bicarbonate and once with 15 mLof water. The organic layer was dried (Na₂SO₄) and concentrated underreduced pressure to give 39 mg (0.08 mmol, 52%) of the4-tert-Butoxycarbonylamino-heptanedioic acidbis-(2,5-dioxo-pyrrolidin-1-yl)ester product (compound 42) as a whitesolid.

Preparation of Di-Osmium t-Boc protected aliphatic linker (Compound 43)

To 20 mg (0.040 mmol) of 4-tert-Butoxycarbonylamino-heptanedioic acidbis-(2,5-dioxo-pyrrolidin-1-yl)ester (compound 42) was added 44 mg(0.064 mmol) of osmium(bPy)₂(histamine)Cl (compound 5), followed by 2 mLmethylene chloride and 0.5 mL DMF. To the reaction mixture 200 μL (1.43mmol) of triethylamine is added and the reaction mixture is allowed tostir at room temperature for 24 to 48 hours as needed for the reactionto complete. The reaction mixture was concentrated under reducedpressure and purified by preparative reverse phase HPLC to give thedi-osmium t-Boc protected aliphatic linker (compound 43).

Preparation of Di-Osmium aliphatic linker (Compound 44)

To 10 mg of di-osmium t-Boc protected aliphatic linker (compound 43) wasadded 1 ml trifluoroacetic acid and allowed to stir at room temperaturebetween 1-2 hours. This was concentrated under reduced pressure to givedi-osmium aliphatic linker (compound 44).

Preparation of biimidazole (Compound 45)

To 25 ml of glyoxal (40 wt % in water) was added 25 mL of water. Thereaction mixture was cool in an ice-bath and ammonia gas was bubbledslowly through the mixture for 7 hours. The reaction mixture wasfiltered to give 710 mg of bidiimdazole (compound 45) as a gray coloredpowder. This was used in the next step without further purification,LC/MS M+H 135.0.

Preparation of dimethyl biimidazole (Compound 46)

To 60 mg (0.44 mmol) of biimidazole was added 1 mL of anhydrous DMF. Thereaction mixture was cooled in an ice-bath and 27 mg (0.67 mmol) of NaH(60% in oil) was added. The reaction mixture was allowed to stir at 0°C. for 1 hour. 140 μL (0.92 mmol) of methyl p-toluene sulfonate wasadded and the reaction mixture was allowed to stir an additional 1 h at0° C. and then two days at room temperature. The reaction mixture wasconcentrated and purified by silica gel column chromatography using 50%ethyl acetate in methanol to give 60 mg (0.36 mmol, 83%) of dimethylbiimidazole (compound 46), LC/MS M+H 163.1.

Preparation of Osmium di biimidazole dichloride (Compound 47)

To 150 mg (0.50 mmol) of OsCl₃ was added 112 mg (0.69 mmol) ofdimethylbiimidazole (compound 46) followed by 280 mg (6.6 mmol) oflithium chloride and 10 mL of anhydrous DMF. The resulting reactionmixture was allowed to reflux under argon atmosphere for 3.5 h andconcentrated. The residue was purified by preparative RP-HPLC to give 95mg (0.15 mmol, 30%) of osmium di biimidazole dichloride (compound 47) asa dark brown powder, LC/MS M+H 586.0.

Preparation of Osmium dibiimidazole hist-t-Boc (Compound 48)

To 50 mg (0.080 mmol) of osmium dibiimidazole dichloride (compound 47)was added 71 mg (0.33 mmol) of [2-(1H-Imidazol-4-yl)-ethyl]-carbamicacid tert-butyl ester (compound 3), followed by 300 μL (2.14 mmol) oftriethylamine and 10 mL of ethanol. The mixture was heated to reflux for18 hours and concentrated. The residue was purified by preparativereverse phase HPLC to give 13 mg (0.016 mmol, 20%) of osmiumdi-biimidazole hist-t-Boc (compound 48) as a dark brown powder LC/MS M+H761.2.

Preparation of Osmium (dimethyl biimidazole)₂ histamine linker (Compound49)

To 4 mg (5.02×10⁻³ mmol) of osmium dibiimidazole hist-t-Boc (compound48) were added 750 μL of methylene chloride and 750 μL oftrifluoroacetic acid. The resulting reaction mixture was allowed to stirat room temperature for 20 minutes and concentrated under reducedpressure. To the residue 5 mL of methylene chloride was added andconcentrated. The addition of methylene chloride and concentrationprocess was repeated three more times and the residue was dried to give3 mg (4.3×10⁻³ mmol, 88%) of osmium (dimethyl biimidazole)₂ histaminelinker (compound 49), LC/MS M+H 661.1.

The osmium (dimethyl biimidazole)₂ histamine linker was prepared as anexample of a mediator that should have a lower redox potential. Lowerredox potentials are of interest for electrochemical assays to avoidinterfering compounds that readily oxidize at higher potentials. Lowermediators are also needed for methods of mixed mediators where the redoxpotential of each mediator needs to be separated by a minimum of 50-100mV to allow independent measurement of each mediator with abipotentiostat as discussed in U.S. Pat. No. 6,294,062. A CV wasperformed with unpurified material of this mediator by dissolving 1 mgof the mediator into 1 ml of PBST for a concentration of about 1.4 mM.The CV indicated that the E_(1/2) potential for this mediator wassignificantly lower that the other mediators prepared. The E_(1/2)potential was about −520 mV vs. Ag/AgCl.

1. A compound of the formula III:

wherein, R and R¹ are the same or different and each can be selected from: 2,2′-bipyridyl, 4,4′-disubstituted-2,2′-bipyridyl, 5-5′-disubstituted-2,2′-bipyridyl, 1,10-phenanthrolinyl, 4,7-disubstituted-1,10-phenanthrolinyl, 5,6-disubstituted-1,10-phenanthrolinyl, or N,N′-dimethyl 2,2′-biimidazole, wherein each substituent is a methyl, ethyl, or phenyl group, and where the R and R¹ groups are coordinated to Os through their nitrogen atoms, R² is a saturated or unsaturated, substituted or unsubstituted, straight or branched chain, hydrocarbyl group having 1-10 carbon atoms; —R³ is H, CH₃ or C₂H₅; L is (CH₂)_(i)Q wherein i is an integer between 1 and 10, and Q is O, S, or NR³; B is a group comprising a ligand capable of binding to a specific analyte binding partner; X is a counter ion; y is selected to provide a neutral salt; and m is from 4-8.
 2. The compound of claim 1 wherein R and R¹ are the same and are selected from 2,2′-bipyridyl, 4,4′-disubstituted-2,2′-bipyridyl, or 5-5′-disubstituted-2,2′-bipyridyl substituted with a methyl, an ethyl or a phenyl group.
 3. The compound of claim 1 wherein L is —(CH₂)_(n)NR³ and n is an integer between 1 and
 10. 4. The compound of claim 1 wherein the substituent R² is a saturated aliphatic group having between 1 and 10 carbons.
 5. The compound of claim 1 wherein B comprises an epitope recognizable by an antibody capable of specific binding to an analyte.
 6. The compound of claim 1 wherein B comprises an epitope capable of binding to an analyte selected from the group consisting of: a biowarfare agent, an abused substance, a therapeutic agent, an environmental pollutant, a protein or a hormone.
 7. The compound of claim 1 wherein L is —(CH₂)_(n)S and n is an integer between 1 and
 10. 8. The compound of claim 1 wherein X is selected from the group of: chloride, bromide, iodide, fluoride, tetrafluoroborate, perchlorate, nitrate, sulfate, carbonate, and sulfite.
 9. A compound of the formula:

wherein, R and R₁ are the same or different and each can be selected from: 2,2′-bipyridyl, 4,4′-disubstituted-2,2′-bipyridyl, 5-5′-disubstituted-2,2′-bipyridyl, 1,10-phenanthrolinyl, 4,7-disubstituted-1,10-phenanthrolinyl, 5,6-disubstituted-1,10-phenanthrolinyl, or N,N′-dimethyl 2,2′-biimidazole wherein each substituent is a methyl, ethyl, or phenyl group, and where the R and R¹ groups are coordinated to Os through their nitrogen atoms; R³ is H, CH₃ or C₂H₅; L is (CH₂)_(i)Q, wherein i is an integer 1-10 and Q is O or NR³; B is a group comprising a ligand capable of binding to a specific analyte binding partner; Z is chlorine or bromine; X is a counter ion; y is selected to provide a neutral salt; and m is 1 or
 2. 10. The compound of claim 9 wherein R and R¹ are the same and are selected from 2,2′-bipyridyl, 4,4′-disubstituted-2,2′-bipyridyl, or 5,5′-disubstituted-2,2′-bipyridyl substituted with a methyl, an ethyl or a phenyl group.
 11. The compound of claim 9 wherein L is —(CH₂)_(n)NR³ and n is an integer between 1 and
 10. 12. The compound of claim 9 wherein Q is NR³.
 13. The compound of claim 9 wherein B comprises an epitope recognizable by an antibody capable of specific binding to an analyte.
 14. The compound of claim 9 wherein B comprises an epitope capable of binding to an analyte selected from the group consisting of: a biowarfare agent, an abused substance, a therapeutic agent, an environmental pollutant, a protein or a hormone.
 15. The compound of claim 9 wherein X is selected from the group consisting of: chloride, bromide, iodide, fluoride.
 16. A method of detecting an analyte in a liquid sample, said method comprising: contacting a portion of said sample a specific binding partner for said analyte and a redox reversible conjugate, said conjugate comprising the compound of claim 18; simultaneously applying a first potential voltage to a first working electrode and a second potential voltage to a second working electrode using a bipotentiostat; and measuring a current generated by a portion of the redox reversible conjugate not bound to the specific binding partner.
 17. A compound of the formula:

wherein, R and R¹ are the same or different and each can be selected from: 2,2′-bipyridyl, 4,4′-disubstituted-2,2′-bipyridyl, 5,5′-disubstituted-2,2′-bipyridyl, 1,10-phenanthrolinyl, 4,7-disubstituted-1,10-phenanthrolinyl, 5,6-disubstituted-1,10-phenanthrolinyl, or N,N′-dimethyl 2,2′-biimidazole wherein each substituent is a methyl, ethyl, or phenyl group, and where the R and R¹ groups are coordinated to Os through their nitrogen atoms, R³ is H, CH₃ or C₂H₅; L is (CH₂)_(i)Q, wherein i is an integer 1-10 and Q is O or NR³; A is —(CH₂)_(j)—NR³, —(CH₂)_(j)—SH, or an activated ester wherein j is an integer between 1-5; Z is chlorine or bromine; X is a counter ion; y is selected to provide a neutral salt; and m is 1 or
 2. 18. The compound of claim 17 wherein A is —(CH₂)_(j)—NH₂ wherein j is an integer between 1-5. 